high-resolution crystal structure of arthrobacter aurescens chondroitin … · high-resolution...

High-resolution Crystal Structure ofArthrobacter aurescens Chondroitin AC Lyase:An Enzyme–Substrate Complex Defines theCatalytic Mechanism

Vladimir V. Lunin1, Yunge Li1, Robert J. Linhardt2, Hirofumi Miyazono3

Mamoru Kyogashima3, Takuji Kaneko3, Alexander W. Bell4

and Miroslaw Cygler1*

1Biotechnology ResearchInstitute, National ResearchCouncil of Canada, andMontreal Joint Centre forStructural Biology, MontrealQuebec, 6100 Royalmount Ave.Montreal, Quebec, CanadaH4P 2R2

2Department of ChemistryDivision of MedicinalChemistry and Department ofChemical and BiochemicalEngineering, The University ofIowa, 115 S. Grand Ave, PHARS328, Iowa City, IA52242-1112, USA

3Central Research LaboratoriesSeikagaku Corporation, Tateno3-1253, Higashiyamato-shiTokyo 207-0021, Japan

4Montreal Proteomics Network740 Dr Penfield Ave., MontrealQuebec, Canada H3A 1A4

Chondroitin lyases (EC 4.2.2.4 and EC 4.2.2.5) are glycosaminoglycan-degrading enzymes that act as eliminases. Chondroitin lyase AC fromArthrobacter aurescens (ArthroAC) is known to act on chondroitin 4-sulfateand chondroitin 6-sulfate but not on dermatan sulfate. Like other chon-droitin AC lyases, it is capable of cleaving hyaluronan.

We have determined the three-dimensional crystal structure ofArthroAC in its native form as well as in complex with its substrates(chondroitin 4-sulfate tetrasaccharide, CStetra and hyaluronan tetrasacchar-ide) at resolution varying from 1.25 A to 1.9 A. The primary sequence ofArthroAC has not been previously determined but it was possible todetermine the amino acid sequence of this enzyme from the high-resolution electron density maps and to confirm it by mass spectrometry.The enzyme–substrate complexes were obtained by soaking the substrateinto the crystals for varying lengths of time (30 seconds to ten hours) andflash-cooling the crystals. The electron density map for crystals soaked inthe substrate for as short as 30 seconds showed the substrate clearly andindicated that the ring of central glucuronic acid assumes a distortedboat conformation. This structure strongly supports the lytic mechanismwhere Tyr242 acts as a general base that abstracts the proton from the C5position of glucuronic acid while Asn183 and His233 neutralize the chargeon the glucuronate acidic group. Comparison of this structure with that ofchondroitinase AC from Flavobacterium heparinum (FlavoAC) provides anexplanation for the exolytic and endolytic mode of action of ArthroACand FlavoAC, respectively.

Crown Copyright q 2004 Published by Elsevier Ltd. All rights reserved.

Keywords: chondroitin lyase; chondroitinase AC; Arthrobacter aurescens;substrate binding; catalytic mechanism*Corresponding author

Introduction

Glycosaminoglycans (GAGs) are the carbo-hydrate components of proteoglycans, which area major component of the extracellular matrix.1

They are highly negatively charged polysacchar-

ides, composed of disaccharide repeating units ofa substituted glucosamine or galactosamineattached through (1,4) linkage to a uronic acidmolecule. These disaccharide units are linked (1,3)or (1,4) into a polysaccharide chain.2 The glucosa-mine/galactosamine units are sulfated extensively,and their synthesis requires the concerted actionof a large number of enzymes.3,4

Glycosaminoglycans are degraded enzymaticallyby two types of enzymes, hydrolases and lyases.5

Hydrolases catalyze cleavage of the glycosyl-oxygen bond by addition of water, producinga saturated disaccharide. Lyases cleave the

0022-2836/$ - see front matter Crown Copyright q 2004 Published by Elsevier Ltd. All rights reserved.

Supplementary data associated with this article can befound at doi: 10.1016/j.jmb.2003.12.071

E-mail address of the corresponding author:[email protected]

Abbreviations used: GAG, glycosaminoglycan; MS,mass spectrometry.

doi:10.1016/j.jmb.2003.12.071 J. Mol. Biol. (2004) 337, 367–386

oxygen–aglycone linkage through proton abstrac-tion, producing an unsaturated disaccharide pro-duct with a double bond between C4 and C5. Theenzymatic mechanisms of hydrolases are wellunderstood and reactions proceed either accordingto the retaining or inverting mechanism.6 On theother hand, the molecular details of the enzymaticmechanism of GAG lyases are still poorly under-stood. A chemically plausible mechanism for the belimination reaction has been proposed;7 however,the constitution of the active site and the roles ofindividual amino acids are not clear. A number ofbacterial species synthesize GAG lyases, enzymesused to degrade and utilize glycosaminoglycansas a source of carbon in the bacterium’s naturalenvironment.5,8 Polysaccharide lyases with knownthree-dimensional structures fall into two architec-tures: the right-handed parallel b-helix (pectate/pectin lyases, chondroitinase B, rhamnoglucuronanlyase) and (a/a)n toroid (n ¼ 5 for Flavobacteriumheparinum chondroitin AC and chondroitin ABClyases, bacterial hyaluronate lyases, xanthan lyase,and n ¼ 6 for alginate lyases). A catalytic mechan-ism has been proposed for pectate lyases, Ca2þ-dependent enzymes,9 but it remains to be seen if itapplies to other lyases having the b-helix topology.Several plausible mechanisms have been proposedfor the lyases with the (a/a)5 toroidal fold witha histidine or a tyrosine residue in the role of ageneral base abstracting the proton from the C5atom of glucuronic acid, and a tyrosine or an argin-ine residue acting as a general acid donating a pro-ton to the bridging O4 atom.10,11 However, there isinsufficient evidence to indicate which of theseproposed mechanisms is utilized by the enzymes.The nature of the group presumed to be necessaryto neutralize the charge of the glucuronic acidcarboxylic group is not clear, since these enzymesdo not require Ca2þ and there is no positivelycharged group in the vicinity of the uronic acid, asexpected from the accepted chemical mechanism.7

Glycosaminoglycan-degrading enzymes withdefined specificity have found widespread appli-cations as analytical tools for the analysis of thestructure of glycosaminoglycans and other poly-saccharides.12 Chondroitin AC lyases are used fre-quently for this purpose. These enzymes cleavethe glycosidic bond on the non-reducing end of anuronic acid and use as a substrate either chondroi-tin 4-sulfate or chondroitin 6-sulfate but notdermatan sulfate. They display a varied degreeof activity toward hyaluronan.13 Enzymes fromtwo sources, chondroitin AC lyase from Arthrobacteraurescens (ArthroAC) and from F. heparinum(FlavoAC), are commercially available (SeikagakuCorporation) and used frequently. The latterenzyme has been cloned and overexpressed inF. heparinum14 and in Escherichia coli.15 We pre-viously determined the three-dimensional struc-ture of this enzyme on its own16 and in complexwith several dermatan sulfate oligosaccharides.10

Here, we present the three-dimensional structureof chondroitin AC lyase from A. aurescens as well

as the complexes with chondroitin tetrasaccharide(DUAp (1 ! 3)-b-D-GalpNAc4S (1 ! 4)-b-D-GlcAp(1 ! 3)-a,b-D-GalpNAc4S, where DUAp is theunsaturated sugar residue, 4-deoxy-a-L-threo-hex-4-enopyranosyluronic acid; GlcAp, glucopyrano-syluronic acid; GalpN, 2-deoxy-2-aminogalacto-pyranose; S, sulfate; and Ac, acetate) andhyaluronan tetrasaccharide substrates.

Despite the purification and characterization ofArthroAC many years ago13,17,18 and its extensiveuse as an analytical tool in glycosaminoglycananalysis, this enzyme has not been cloned and itsamino acid sequence has not been determined,although its amino acid composition and carbo-hydrate content were reported.18 We obtainedcrystals that diffract up to 1.25 A resolution. Thehigh-resolution data led to high-quality electrondensity maps of native enzyme and several com-plexes, and allowed us to deduce confidently theamino acid sequence for 99% of the amino acidresidues and to propose the molecular details ofthe catalytic mechanism, which is common toFlavoAC and hyaluronate lyases. Here, we followthe nomenclature introduced by Davies et al.19 anddesignate the sugars on the reducing end of thebreak with a þ sign and the sugars on the non-reducing end with a 2 sign. In this nomenclature,the enzymes break the bond between sugars 21and þ1, the latter being an uronic acid.

Results and Discussion

Amino acid sequence and its conservation

The molecular mass of the entire molecule wasmeasured by ion spray mass spectrometry. Twospecies were present with molecular masses of79,502 Da and 79,840 Da. The greater mass corre-sponds well to the molecular mass of 79,785 Da(average mass) calculated from the amino acidsequence. Based on the analysis of MS/MS spectra,we believe that the smaller mass corresponds tothe fragment missing three N-terminal amino acidresidues. Such a good agreement between the pre-dicted and measured molecular mass indicatedthat (1) no major assignment errors were madeand (2) no glycosylation or other modificationwere present (FlavoAC is glycosylated).16

Peptides extracted from an in-gel trypsin digestof purified A. aurescens chondroitin AC lyase wereanalyzed by LC-QToF mass spectrometry asdescribed in Materials and Methods. The resultingpeaklist of fragmentation spectra was matched in-house against the sequence deduced from the crys-tal structure employing Mascot (MatrixScience)software.20 Matched tandem MS spectra were con-firmed manually and the remaining spectra wereinterpreted manually. This process was repeatedseveral times, using the two sets of experimentaldata iteratively to determine the optimal sequenceof the lyase (Supplementary Material).

The results of MS/MS analysis of 202 tandem

368 Chondroitinase AC Crystal Structure and Mechanism

mass spectra covering 88.5% of the entireArthroAC sequence (see Materials and Methods)confirmed the amino acid sequence identifiedfrom electron density maps. Moreover, this analy-sis of individual MS/MS fragmentation dataallowed us to make side-chain assignments forseveral residues located in flexible loops for whichelectron density was not easily interpretable(shown in small letters in Figure 1).

The agreement for 18 (4–21) residues betweenthe residue type derived from the electron densitymaps and mass spectrometry, and that determinedby Edman degradation provides an independentmeasure underscoring the low level of errors to beexpected in our assignment. While the sequence ofArthroAC has not been determined previously, itsamino acid composition was reported nearly 30years ago.18 These data showed good correlationwith the amino acid sequence of ArthroAC in thiswork, supporting our assignments (Table 1).

The derived sequence of ArthroAC was used toidentify homologous sequences in the NCBI data-base with the program BLAST.21 There are ,50such sequences for which the similarity extendsalong the entire protein. They include hyaluronate,xanthan and chondroitin AC lyases. ArthroACshows the highest level of sequence identity withvarious hyaluronan and xanthan lyases (38%) anda lower level of identity with chondroitin AClyase from F. heparinum (24%).

The structures of four proteins representative ofthese sequences are known; namely, chondroitin AClyase 1CB8,16 Streptococcus pneumoniae hyaluronatelyase 1EGU,11 Streptococcus agalactiae hyaluronate

lyase 1F1S22 and Bacillus sp. xanthan lyase 1J0M.23

Structure-based alignment of their sequences isshown in Figure 1. The residues important for theintegrity of the active site (see below) includeAsn183, His233, Tyr242, Arg296 and Glu407 ofArthroAC and are conserved in all of the relatedenzymes, and Glu412 is replaced by an aspartateresidue in one case.

The enzyme chondroitin ABC lyase I showsgood sequence similarity to the above-mentionedenzymes only for the C-terminal domain. How-ever, its three-dimensional structure (PDB code1HN0) showed that the catalytic domain has (a/a)5

topology and can be structurally aligned with thecatalytic domains of the other lyases.24 While thesubstrate-binding site shows little sequence con-servation, the active-site residues are conserved,suggesting the same enzymatic mechanism. Thereis no equivalent, however, to the Asn183 ofArthroAC, and there are differences in the localstructure in this region. Huang et al. suggestedthat this enzyme utilizes as a replacement an argin-ine residue remote in the linear sequence.24 Thestructure-based alignment of chondroitin ABClyase I with the other lyases is included in Figure 1.

Overall fold

The ArthroAC molecule has an overall a þ barchitecture and consists of two domains (Figure 2).The N-terminal a-helical domain contains 13a-helices, ten of which form an incompletedouble-layered (a/a)5 toroid as classified withinthe SCOP database.25 There is a long, deep grooveon one side of the toroid that forms the locationof the active site and substrate-binding site. Threea-helices at the N terminus precede the (a/a)5

toroid and constrict the cleft on one side. Residuesconserved across the sequences of proteins homo-logous to ArthroAC cluster in the area of this cleft.The C-terminal domain is composed almostentirely of antiparallel b-strands arranged intofour b-sheets. The first two sheets contain nineb-strands, some of them rather long. The thirdsheet has seven b-strands and the last one fiveb-strands. There is only one short a-helix withinthis domain (Figure 2). The second domain can besubdivided into two subdomains; the first encom-passes the first two large b-sheets and one shorta-helix, while the second subdomain is composedof the third and fourth b-sheet.

Substrate-binding site

Initial experiments of soaking native crystalsof ArthroAC in a 5 mM solution of chondroitin4-sulfate tetrasaccharide substrate for prolongedtimes before collecting diffraction data showedclear density for only a disaccharide product, indi-cating that, like other GAG lyases,10 ArthroACretains enzymatic activity in the crystals. Therefore,we decided to investigate by X-ray diffractionthe enzyme–substrate complex as a function of

Table 1. Comparison of percentage distribution of aminoacids between chemical amino acid analysis and crystal-lographic assignment

Residuetype

Sequenced from the map(%)

From Hiyama &Okada18

Ala 14.1 13.4Arg 5.2 4.7Asx 9.1 9.0Glx 6.35 6.5Trp 2.5 2.4Val 7.0 8.2Ser 6.7 6.6Thr 8.85 8.4His 1.85 1.7Phe 2.9 2.9Gly 10.6 11.9Pro 4.0 3.3Ile 3.6 3.3Leu 9.1 8.8Lys 3.3 3.3Tyr 2.5 2.6Met 1.45 1.1Cysa 0.9 2.3

a The significant difference in the number of cysteine residuesbetween our data and those reported by Hiyama & Okada whilethere is very good agreement for the other amino acids, is likelyrelated to the fact that hydrolysis was used for the determi-nation of most amino acids while sulfhydryl titration was usedfor cysteine.

Chondroitinase AC Crystal Structure and Mechanism 369

Figure 1 (legend opposite)

soaking time. The soaking time ranged from 30seconds to ten hours (Tables 2 and 3). At the endof each soak, the crystal was immediately flash-frozen and diffraction data collected (resolutionvarying between 1.25 A and 1.6 A, Table 3). Hya-luronan tetrasaccharide was also used as a sub-strate with a soaking time of two minutes anddiffraction data were collected to 1.9 A resolution.The structures were refined independently. Ineach case, the ArthroAC molecule was refinedfirst, then the difference electron density map wasinspected and interpreted appropriately. Themodeled substrate/product was included in therefinement. Several sugar units were clearly visiblein each refined model. The relative occupancy ofthe 2 and þ sites along the timed snapshots wereevaluated as described in Materials and Methods.The reaction in the present crystals occurs on theminute timescale, indicated by well-defined elec-tron density for the entire tetrasaccharide substrateafter 30 seconds and even longer soaks (Figure 3(a)and (b); and Table 4). Indeed, the substrate is bestdefined in this dataset, with assigned occupanciesof ,0.6. Somewhat higher occupancy for the (21,

22) subsites suggests a partial presence of thedisaccharide product in the 2 sites. For the tenhour soak, the sugar units in subsites (21, 22) areclearly visible in the difference electron densitymap and refine with an occupancy of 1.0, whilethe derived occupancy for the sugars at the (þ1,þ2) subsites were 0.25. In the complex ofArthroAC with hyaluronan tetrasaccharide, onlythe sugar units in subsites (21, 22) were visiblein the electron density, corresponding to a disac-charide reaction product and in accord with higheractivity of ArthroAC toward hyaluronan.13 Thelocation and orientation of the hyaluronan sugarsis the same as the corresponding sugars of thechondroitin sulfate tetrasaccharide substrate. Inthe following discussion, we refer to the ArthroAC–tetrasaccharide complex after a 30 seconds soak.

The oligosaccharide is bound within the groovein the N-terminal domain (Figure 2) and makescontacts with residues Asn124, Trp125, Trp126,Arg134, Gln169, Arg174, Asn183, His233, Tyr242,Arg296, Arg300, Asn303, Asn410 and Trp465(Figure 3(c)). Tryptophan residues play an essentialrole in substrate binding. Two of these residues,

Figure 1. Structure-based sequence alignment for ArthroAC, FlavoAC (1CB8), S. pneumoniae hyaluronate lyase(1EGU), S. alagalactiae hyaluronate lyase (1F1S), Bacillus sp. xanthan lyase (1J0M) and P. vulgaris chondroitin ABClyase I (1HN0). Insertions in the ArthroAC sequence that close off one end of the substrate-binding site are boxed,a-helices are marked in white letters on black background and b-stands are marked by black letters on gray back-ground. The secondary structure assignments follow sPDBv.51 Residues conserved in all six proteins are marked byan asterisk (*) above the sequence. Arrows mark residues essential for catalysis, Asn183, His233, Tyr242 and Arg296and Glu407. Several residues that were assigned on the basis of the MS/MS data alone (disordered side-chain) areshown in small letters. Letter ‘x’ indicates (Glu/Gln) or (Asp/Asn), which we cannot distinguish, and questionmarks (?) indicate residues for which we are less certain of their amino acid type.

Figure 2. Stereo drawing of the ribbon representation of ArthroAC showing the bound tetrasaccharide. The individ-ual a-helical hairpins of the N-terminal (a/a)5 toroid are in different colors. The individual b-sheets of the C-terminaldomain are in discrete colors. Insertions in ArthroAC blocking the cleft are in gray. The substrate is shown in stickrepresentation and colored in magenta. The Figure was prepared with the programs MOLSCRIPT52 and Raster3d.53


Trp126 and Trp465, provide stacking interactionswith the sugar units occupying positions 21 andþ2, respectively, while Trp125 is aligned edge-onand forms a hydrogen bond with the bridgingoxygen atom between the þ2 and þ1 units. The4-O-sulfo groups of the substrate form severalinteractions with the protein. The 4-O-sulfo groupof the þ2 sugar makes H-bonds to Gln169 and,

through a bridging water molecule, to Asp222 andGln232. The 21 sugar 4-O-sulfo group is posi-tioned just above the guanidinium group ofArg300 and, in addition, forms H-bonds to Glu412and Asn598 through a bridging water molecule.Both 4-O-sulfo groups contribute to substrate bind-ing but do not add significantly to the specificity ofsubstrate recognition.

The chondroitin sulfate tetrasaccharide used inthese studies was obtained by the action of GAGlyases and contained an unsaturated ring at thenon-reducing end, with a C4vC5 double bond.10

The electron density for the 22 sugar correspondsvery well to this unsaturated ring in E1 confor-mation, with the C5 having sp2 hybridization(Figure 3a). A list of hydrogen bonds between thetetrasaccharide and the protein side-chains isgiven in Table 5.

Most interesting from the viewpoint of themechanism of catalysis is the conformation andinteractions with the enzyme of the þ1 glucuronicacid. The electron density shows that this ringassumes a distorted boat conformation O,3B with

Table 3. Refinement statistics

Model

Hgderivative Native

CStetra

30 secondsCStetra

10 minutesCStetra

10 hoursHAtetra

Two minutes

Resolution range 50–1.3 50–1.35 50–1.45 50–1.5 50–1.25 50–1.9R-factor (Rfree) 0.134 (0.155) 0.130 (0.175) 0.138 (0.177) 0.136 (0.170) 0.113 (0.142) 0.190 (0.251)No. non-hydrogen protein atoms 5687 5623 5617 5627 5646 5629No. of water molecules 1103 1025 1049 1061 1107 837Average B-factor (A2)Protein main-chain atoms 14.9 14.1 15.5 15.4 13.6 21.8Side-chain atoms 16.5 16.3 17.9 16.7 15.4 22.4Water molecules 28.7 29.4 31.4 30.5 29.6 31.6Substrate atoms 24.8 21.2 15.5 22.5r.m.s.d. bond length (A) 0.019 0.022 0.023 0.021 0.021 0.018r.m.s.d bond angle (deg.) 1.84 1.97 1.84 1.86 2.01 1.72Ramachandran plot. Residues in:Most favorable region (%) 90.8 90.7 90.5 89.7 89.9 88.3Disallowed regions (%) 0.3 0.3 0.3 0.3 0.3 0.5

Table 2. Data collection statistics for various substrate soaking times

Soaking timeSubstrate

CStetra HAtetra

30 secondsTwo

minutesTen

minutes 35 minutes Two hours Four hours Ten hoursTwo

minutes

Wavelength (A) 0.9798 0.9798 0.9798 0.9798 0.9798 0.9798 0.9798 0.9798a (A 57.9 57.6 57.7 57.6 57.6 57.6 57.6 57.6b (A) 86.9 86.5 86.4 86.4 86.5 86.5 86.3 86.3c (A) 81.5 80.7 80.6 80.5 80.6 80.6 80.5 80.6b (deg.) 107.0 106.8 106.9 106.9 106.9 106.9 107.0 106.9Resolution range(last shell)

50–1.41(1.46–1.41)

50–1.6(1.66–1.6)

50–1.5(1.55–1.5)

50–1.35(1.4–1.35)

50–1.3(1.35–1.3)

50–1.35(1.4–1.35)

50–1.25(1.29–1.25)

50–1.9(1.97–1.9)

Rsym (last shell) 0.081(0.794)

0.077(0.739)

0.077(0.483)

0.055(0.567)

0.057(0.517)

0.059(0.530)

0.058(0.430)

0.075(0.652)

Completeness (%)(last shell)

99.9 (99.9) 100 (100) 99.9 (99.9) 98.3 (96.3) 96.5 (89.1) 96.1 (93.5) 90.8 (56.4) 100 (99.9)

I=sðIÞ (last shell) 8.1 (2.0) 8.3 (2.5) 9.2 (3.4) 8.4 (2.0) 11.1 (3.5) 8.9 (2.9) 9.8 (2.8) 7.7 (2.8)Total reflections 706,542 501,855 460,480 452,905 1,338,199 610,947 939,404 235,444Unique reflections 146,507 100,158 120,769 162,044 178,855 158,853 188,554 60,174Redundancy 4.8 5.0 3.8 2.8 7.5 3.8 5.0 3.9

Table 4. Relative occupancies of sugars in positions 22,21, þ1, þ2 for different soaking times

Soak timeSite occupancy

22 21 þ1 þ2 Phosphate

Native 1.0CStetra 30 seconds 0.7 0.7 0.6 0.6 0.4

Two minutes 0.7 0.7 0.5 0.5 0.5Ten minutes 0.7 0.7 0.4 0.4 0.635 minutes 0.7 0.7 0.4 0.4 0.6Two hours 1.0 1.0 0.4 0.4 0.6Four hours 1.0 1.0 0.3 0.3 0.7Ten hours 1.0 1.0 0.25 0.25 0.7

HAtetra Two minutes 1.0 1.0 – – 1.0


the hydroxyl groups equatorial and the C5carboxylate group pseudoaxial (Figure 3(b)). Thiscarboxylate group is placed exactly opposite theconserved side-chain of Asn183, whose OD1 andND2 atoms are clearly distinguished by their peakheight in the electron density map (SupplementaryMaterial). The distance between the amide ND2atom and the carboxyl O6A is 3.1 A, and thatbetween carbonyl OD1 and carboxyl O6B is 2.6 A.This short O6B· · ·OD1 distance suggests the exist-ence of a hydrogen bond between them,26 whichin turn would indicate that the glucuronic acidcarboxylate group is protonated and therefore in aneutral state. The O6A, in addition to accepting ahydrogen bond from ND2 of Asn183, also forms asecond, 2.8 A long hydrogen bond with the NE2atom of His233. The geometry of hydrogen bondsinvolving the carboxylate group is very close toideal, the C6–O6A/B-donor angles are in therange 115–1248 and the –COO group and thethree hydrogen bond donor atoms are nearlycoplanar (Figure 3(d)). His233 donates, in addition,a second hydrogen bond from the ND1 atom tothe side-chain of the conserved Glu407; therefore,this histidine residue must be protonated. Thehydroxyl groups at C2 and C3 of the þ1 unit areheld firmly through several hydrogen bonds.Atoms O2 and O3 are H-bonded to OD1 and ND2of Asn124, respectively, while O2 is H-bondedalso to ND2 of Asn410 (Figure 3(c)).

Two other residues make crucial contacts withthe substrate. The hydroxyl O atom of Tyr242 iswithin H-bonding distance of the bridging oxygenatom between þ1 and 21 sugars (2.9 A), and is3.3 A from O5 of the þ1 sugar ring. This hydroxyl

group is also only 2.8 A from the C5 of the glucu-ronic acid, with the O atom nearly along thederived direction of the C5–H5 bond (Figure3(d)). The Arg296 side-chain also forms a hydrogenbond to the bridging oxygen atom between þ1 and21 sugars and is 3.0 A from the Tyr242 hydroxylgroup. On the other hand, the distance from thepotential base His233 NE2 to the C5 of the þ1glucuronic acid is relatively long at 4.0 A.

The electron density corresponding to thecarboxylate group of the glucuronic acid in the þ1site showed not two but three bulges, with thethird one having somewhat lower density. Thisshape was common to the density observed in alldatasets and its position coincided with the phos-phate group in the native structure. We havemodeled a phosphate group with partial occu-pancy in the same location (Table 4), assumingthat it is present there when the glucuronic aciddoes not occupy this site. The total occupancy ofthe þ1 site, that is glucuronic acid plus phosphate,equals 1.

A strong peak in the electron density map wasfound in the proximity of the tetrasaccharide. It issurrounded by six oxygen atoms in tetragonalbipyramidal coordination, with distances to theequatorial oxygen atoms of 2.2–2.4 A and to theaxial oxygen atoms of 2.8 A. This peak was inter-preted as a sodium ion. The equatorial ligands arethe carbonyl groups of His233 and Trp465 andtwo water molecules, while the axial ligands arethe OG1 of Thr235 and a water molecule.

Substrate specificity

The initial characterization of ArthroAC showedthat the enzyme degrades chondroitin 4-sulfate,chondroitin 6-sulfate and hyaluronan.13 Our resultswith soaking various oligosaccharides indicatedthat ArthroAC displays higher activity towardhyaluronan than to chondroitin sulfate. We havedetermined the kinetic parameters of ArthroACwith GAG obtained from whale cartilage (CS-A,predominantly chondroitin 4-sulfate), shark carti-lage (CS-C, predominantly chondroitin 6-sulfate),shark fin (CS-D, a mixture of chondroitin 4-sulfate,6-sulfate and -4,6-disulfate), low molecular masshyaluronan and high molecular mass hyaluronan(Table 6). Indeed, the Vmax for hyaluronan is twiceas high as that for chondroitin sulfate, while theKM calculated on a per monomer basis is aboutthree times lower. These values point to a rathersmall contribution made by the sulfate groups tothe total binding energy of the substrate.

Comparison with other GAG lyases

ArthroAC is very similar in overall structure toother GAG lyases with the (a/a)5 fold. The closestsimilarity is with S. pneumonia hyaluronate lyase(SpHL, PDB code 1EGU). These two structuressuperimpose with root-mean-squares (rms) devi-ation of 1.3 A for 632 Ca atoms out of 750 residues

Table 5. Close contacts between the substrate and theprotein in CStetra complex for the 30 second soakexperiment

Sugar number Atom Protein atom Distance (A)

22 O2 OD2 Asn303 2.85O2 NH1 Arg300 3.07O3 OD1 Asn303 3.03O6A NH1 Arg134 3.01O6B NH2 Arg134 2.90

2 1 O7 NH2 Arg300 2.92O7 NH1 Arg300 3.01SO43 NE Arg300 3.27SO44 NH2 Arg300 3.46

þ 1 O2 ND1 Asn410 3.20O2 OD1 Asn124 2.77O3 ND2 Asn124 2.91O4 NH2 Arg296 3.02O4 OH Tyr242 2.88C5 OH Tyr242 2.75O6A NE2 His233 2.76O6A ND2 Asn183 3.11O6B OD1 Asn183 2.62

þ 2 O1 NH2 Arg174 3.22O3 NE1 Trp125 3.32O7 NH1 Arg174 3.07SO41 NE2 His233 2.85SO43 NE2 Gln169 2.34


(Figure 4). The superposition with FlavoAC resultsin an rms deviation of 1.4 A for 468 Ca atoms. Thesimilarity extends as well to the general mode ofsubstrate binding. Comparison with the SpHL

Y408F mutant (inactive, equivalent to Y242 ofArthroAC) complexed with hyaluronan oligo-saccharide,27 FlavoAC complexed with the derma-tan sulfate hexasaccharide, and its inactive Y234F

Figure 3 (legend opposite)


mutant (ArthroAC Y242 equivalent) complexedwith chondroitin tetrasaccharide10 shows that themode of oligosaccharide binding is nearly identi-cal, with the largest difference restricted to theglucuronic acid at the þ1 site (Figure 4(b)). Theside-chains making crucial contacts with theoligosaccharide are conserved in their type andposition. In the previously observed complexeswith Tyr-to-Phe mutant enzymes, the þ1 glucuro-nic acid ring is in a chair conformation, with thecarboxylic group at C5 in an equatorial position.

That differs from the conformation of the glucuron-ate sugar observed in the wild-type ArthroAC–substrate complex, where the ring forms a dis-torted boat with a pseudoaxial carboxylate group.

A detailed comparison of these structuresreveals subtle differences in the hydrogen bondingnetwork involving active-site residues. In ArthroAC,the hydrogen bonds between the C5 carboxylicgroup of the þ1 sugar and Asn183 and His233have nearly ideal geometry. The correspondingH-bonds in the FlavoAC(Y234F) and SpHL(Y408F)

Figure 3. (a) Electron density for the tetrasaccharide of chondroitin 4-sulfate substrate in the omit map calculatedwithout the substrate present with the data for the 30 seconds soak of ArthroAC in 5 mM tetrasaccharide solutionand contoured at the 3s level. The phosphate group with partial occupancy is shown. An arrow marks the bond thatis cleaved by the enzyme. (b) Close-up of the same map for the þ1 glucuronic acid showing the distorted boat confor-mation; (c) stereo view of the substrate-binding site. The tetrasaccharide is shown in thick semitransparent lines,hydrogen bonds are shown with broken lines; (d) close-up of the active site showing residues involved in catalysisand the hydrogen bonding network connecting these residues. Magenta dotted line marks the close contact betweenthe Tyr hydroxyl group and the C5 atom of glucuronic acid.


complexes have less favorable geometry (Figure4(b)). This asparagine residue in SpHL is modeledwith the opposite orientation of the amide groupto that in the other two enzymes. In the FlavoAC(Y234F)-chondroitin sulfate tetrasaccharide com-plex, the glucuronate sugar remains in a chair con-formation with all substituents to the ring beingequatorial, but rotates so that the carboxylic groupoccupies the space vacated by the missinghydroxyl group of Tyr234.10 Thus, even smallchanges in the active site affect the mode of bind-ing of the substrate and may result in a non-productive binding.

Catalytic mechanism

The enzymatic reaction carried out by GAGlyases is thought to proceed via abstraction of theC5 proton by a general base followed by protondonation by a general acid or a water molecule tothe bridging O4, with concomitant b-eliminationof the leaving group.7 Recent kinetic analysis ofthe FlavoAC using a well defined synthetic sub-strate agrees with the predicted stepwise, asopposed to concerted, mechanism.28 A proposalfor the mechanism of polysaccharide lyases formu-lated by Gacesa included the neutralization of theacidic group by a positively charged group to shiftthe equilibrium toward the enolate tautomericform.7 Unlike polysaccharide lyases that adoptthe b-helix fold, the structures of FlavoAC andhyaluronate lyases showed an absence of such apositively charged group in the vicinity of theacidic group of uronate. Instead, an asparagineside-chain was found to face and form a H-bondwith the acidic group, leaving the issue of neutral-ization of the acidic group an open question. Thestructures of the complexes presented here suggestthat Asn183 OD1 forms a strong hydrogen bondwith this carboxylic group, substantially increasingits pKa and promoting its protonation.29,30 Thisasparagine residue is aided by His233, which also

forms a H-bond to the carboxylate group of uronicacid and is protonated in the complex, as judgedfrom it being hydrogen bonded to two acidicgroups (Figure 3(d)).

On the basis of structural evidence from GAGlyase–oligosaccharide complexes, several propo-sals have been put forward as to the identity ofthe general base and general acid participating inthe reaction. Jedrzejas and co-workers proposedthat the proton is abstracted by a nearby histidineresidue (His233 in the present structure), and thatanother proton is donated to O4 by a tyrosine resi-due (Tyr242 here).11,27,31 They support this pro-posed role of the histidine residue as the generalbase by the close distance between the histidineNE1 and C5 in their structures. Huang et al. con-sidered three possible mechanistic scenarios, ulti-mately favoring one in which a tyrosine residueinitially functioned as a general base and subse-quently as a general acid.10 In a structurally relatedbut sequence-distant alginate lyase also, the centralcatalytic role was assigned to a tyrosine residue.32,33

The kinetic characterization of FlavoAC with asynthetic substrate strongly favors tyrosine as theproton acceptor.28

We have carefully re-analyzed the availablestructural data to assess the role of histidine(His233 in ArthroAC and its equivalent) in cataly-sis. The first suggestion for the role of histidine asa general base was derived from the structure ofhyaluronan lyase and its complexes with a disac-charide product,11,34 in which the þ1 site was notoccupied. These authors modeled the þ1 sugarand estimated the NE1· · ·C5 distance to be ,4 A.This value corresponds to the N· · ·C van derWaals distance and seems to be too long for theproposed proton-abstracting role of the histidine.

A direct observation of the enzyme–substratecomplex was accomplished for FlavoAC(Y234F)10

and for SpHL(Y408F).27 In the case of FlavoAC(Y234F), it was concluded that the mode of sub-strate binding in the þ1 site is influenced by the

Table 6. Kinetic parameters of ArthroAC

CS-A CS-C CS-D LMHA (50K) HA (1000K)

V (DABS/s) 1.42 £ 10 2 3 1.74 £ 10 2 3 6.10 £ 10 2 4 2.57 £ 10 2 3 2.78 £ 10 2 3Km (mg/ml) 0.196 0.196 0.188 0.052 0.082Ma (Da) 505.2313 511.1607 532.166 401.3 401.3Km (mM) 0.387 0.383 0.353 0.130 0.205Mb (Da) 19,000 43,000 30,000 50,000 1,000,000Km (nM) 10.304 4.554 6.256 1.046 0.082

Disaccharide composition (%)c

GAG source Whale cartilage Shark cartilage Shark finDDi-0S 1.6 1.7 0.6DDi-6S 19.3 72.9 43.9DDi-4S 76.2 15.4 26.9DDi-diSD 2.7 9.3 21.3DDi-diSE 0.3 0.6 7DDi-triS – – 0.3

a Disaccharides composition.b GPC-HPLC (CS STD).c Analysis of unsaturated disaccharides from glycosaminoglycan by HPLC.


mutation and does not reflect the reaction inter-mediate (the C5 carboxylic group occupies thevolume vacated by the missing Tyr hydroxylgroup in the Phe mutant) therefore precluding thedistinction between the possible mechanisms. Inthe case of SpHL(Y408F), the distance betweenHis399 and the C5 of the uronic acid is 3.73 A, butthe C5 proton lies almost along the line of CD2–NE2 bond, very poor geometry indeed for protonabstraction. If the Phe408 is replaced in this modelby the original Tyr, its hydroxyl O atom would beonly ,3.0 A from C5 and would have a muchbetter geometry for interacting with the C5 proton.Hence, we find these results equally inconclusiveconcerning the assignment of His as the generalbase.

A structure of the crystals of S. agalactiae hya-luronate lyase soaked for several days in 10–50 mM hexasaccharide substrate was reportedrecently.31 In the model, the enzyme assumes amore open conformation and Tyr408 and His399are further away from the carbohydrate. Specifi-cally, the distance of 5.7 A between the NE1 atomof His399 and the C5 atom of the þ1 sugar doesnot allow the conclusion to be drawn that the Hisplays the role of general base. As well, the temper-ature factors in this structure (PDB code 1LXM)for most of the atoms of the modeled substrate areequal to 100 A2, significantly higher than the aver-age value of ,35 A2 for the surrounding atoms,suggesting poor order of the substrate. The differ-ence electron density map (not reported in the

Figure 4. (a) Stereo view of the superposition of the Ca traces of ArthroAC (blue) and SpHL (1EGU) (red); (b) overlayof oligosaccharide substrates from ArthroAC (blue), FlavoAC(Y234F) (1HMW, magenta) and SpHL(Y408F) (1LXK,green) based on the superposition of the backbone of active site Asn, His, Tyr (Phe) and Arg residues. The Figurewas prepared with programs sPDBv51 and POV-Raye (http://www.povray.org/).


original paper) calculated from the depositedstructure factors and coordinates, with the sub-strate excluded from calculations, showed weakand scattered density that in our view cannot bemodeled reliably as a single carbohydrate molecule(Supplementary Material). The role of His399 as ageneral base is further put in doubt by the factthat His399Ala mutant of SpHL retains a signifi-cant fraction (6%) of the wild-type enzymeactivity.11

The present series of timed snapshots ofstructures of complexes of the wild-type ArthroACwith chondroitin 4-sulfate tetrasaccharide substrateprovides very strong support for Tyr242, rather

than His233, playing the role of a general base inthe first step of catalysis. The proposed reactionmechanism is shown in Figure 5. Substrate bindingto ArthroAC is associated with a deformation ofthe glucuronate sugar ring at the þ1 site from achair to a distorted boat and protonation of itsacidic group. Such a change to a higher-energy con-formation of the sugar ring is not unusual and hasbeen observed in other carbohydrate-processingenzymes.35 – 40 The distortion of the ring brings thepseudoaxial acidic group coplanar with the amidegroup of Asn183, and the presence of an additionalproton leads to the formation of two hydrogenbonds, one of them being a strong O· · ·H· · ·O

Figure 5. Proposed catalytic mechanism of (a/a)5 GAG lyases. Panel 1, Tetrasaccharide bound in the substrate-bind-ing site. The hydrogen bonding network involving the active site and substrate-binding residues is shown schemati-cally. The tryptophan residues stack against sugars in 21 and þ2 subsites. His233 is protonated and OD1 of Asn183forms a strong hydrogen bond with the protonated carboxylic group of glucuronic acid. Deprotonated Tyr242 abstractsthe C5 proton. Panel 2, Tyr242 accepts the proton from C5 atom leading to carbanion formation. Now Tyr242 formshydrogen bond with bridging O4. Panel 3, The proton from Tyr242 is transferred to the O1 of galactosamine in the21 subsite with concomitant break of C4–O4 bond and formation of unsaturated ring in þ1 subsite. Panel 4, Move-ment of Trp465 triggers the release of disaccharide product from (þ1, þ 2) subsites, reorganization of the active siteand release of product from (22, 21) subsites. The enzyme is ready for the next catalytic cycle.


hydrogen bond. The high-resolution structure ofthe complex shows that the Oh atom of Tyr242 ispositioned 2.8 A from the C5 atom of the þ1 glu-curonic acid, along the direction of the C5–Hbond. This Oh atom is at the same time withinhydrogen bonding distance of the O atom bridgingþ1 and 21 sugars and of Arg296. We postulatethat Tyr242 becomes deprotonated upon substratebinding and that the Oh takes up the proton fromthe C5 atom of glucuronic acid (Figure 5, panel 2),which is then transferred to the bridging O atom2.9 A away, with a concomitant break of the O4–C4 bond (Figure 5, panel 3). A similar mechanismutilizing a deprotonated tyrosine residue was pro-posed for alginate lyase A1-III,33 which is structu-rally similar to ArthroAC despite little sequencesimilarity.

What then is the role of His233? This side-chain,and its equivalents in other lyases, is in theproximity of the þ1 glucuronic acid, but with theNE2 atom at a distance of ,4 A or more from C5of the þ1 sugar, typical for van der Waals inter-actions. The geometry of the H· · ·NE2 is also suchthat the direction of the expected lone pair on NE2is ,608 away from the direction to the C5 proton.This geometry is observed consistently in the struc-tures of the FlavoAC and SpH carbohydrate com-plexes. Finally, NE2 of His233 is hydrogen bondedto the carboxylate group of the þ1 glucuronic acidand, thus, must be protonated. Considering thesefacts collectively, it is unlikely that His233 playsthe role of general base to remove the C5 proton.We postulate that this side-chain has two functions:(1) it helps properly orient the carboxylate group ofthe glucuronic acid through the NE2· · ·O6A hydro-gen bond; (2) being protonated in the complex andin conjunction with Arg296, it lowers the pKa ofTyr242 leading to the deprotonation of its hydroxylgroup and priming it for the role as a general base(Figure 5, panel 1). This second function of His233is directly related to the nearby presence ofGlu407, which engages the histidine ND2 protonin a hydrogen bond and through electrostatic inter-actions aids in histidine protonation. Glu407 formsat the same time a salt-bridge with Arg296, com-pleting a tetrad of hydrogen bonded residues(Tyr242, His233, Arg296, Glu407) forming theactive site.

The conservation in FlavoAC and hyaluronatelyases of all Asn183, His233, Tyr242, Arg300 andGlu407, residues critical for the above describedmechanism supports the view that this is a com-mon catalytic mechanism for this class of enzymes.

A possible mechanism of product release wasilluminated by the structure of thimerosal-soakedArthroAC solved here. Of the three bound heavy-atoms, two bound to surface-exposed cysteine resi-dues. The third bound to Cys408, which in thenative structure is covered by the 460–469 loopand inaccessible to the solvent, suggesting thatthis loop is flexible enough to allow access for arelatively large thimerosal molecule. At the tip ofthis loop is Trp465, which provides stacking inter-

actions to the sugar unit of the substrate bound inthe þ2 site (Figure 3(c)). This loop in ArthroAC-Hg assumes an open conformation, indicating itsintrinsic mobility even in the crystal environment.When Trp465 is sequestered from the substrate-binding cleft, the side-chains of Arg296 andHis233 extend into the volume previously occu-pied by Trp465 (Figure 6). His233 moves awayfrom Glu407, forming hydrogen bonds with watermolecules, and is most likely neutral. The salt-bridge between Glu412 and Arg296 is broken, butthe side-chain of Glu407 follows Arg296 andforms a stronger salt-bridge, with two H-bonds.Movement of this loop after the reaction is com-pleted would substantially decrease the binding ofthe product in (þ1, þ2) sites and aid in its releasefrom the enzyme. A rearrangement of residues inthe active site together with their hydrogen bond-ing network and neutralization of His233 wouldlead to reprotonation of Tyr242 making it readyfor the next catalytic cycle (Figure 5, panel 4). Wepropose that the deprotonation of Tyr242 and pro-tonation of His233 is concomitant with substratebinding and triggered by the interaction of thecarboxylate group on the þ1 sugar with Asn183and His233.

The access of the substrate to the active sitenecessitates either local movements of two tothree loops closing off the binding site,10 or ahinge movement between the domains leading toa global movement of the N and C-terminaldomains creating an opening to the active site.27,33

Structural rationale for the exolytic versusendolytic mode of action

Enzymatic characterization of ArthroAC showedthat it acts as an exolyase, releasing disaccharidesfrom the glycosaminoglycan substrate,41 whileFlavoAC acts as an endolyase.42 Comparison ofthe structures of these two enzymes provides arationale for the observed differences in themode of action. ArthroAC has two insertions inthe N-terminal domain relative to the FlavoAC: ,15residues (Arg23–Ser38) and 25 residues (Thr343–Gly366). These two segments form a-helices-containing loops near the N and C termini of thedomain, which come together, closing off a largepart of the cleft running along the side of the (a/a)5

toroidal N-terminal domain. These loops form awall that converts the open cleft into a deep cavityand precludes binding of an extended oligosac-charide. The size of the cavity restricts the bindingof the carbohydrate on the non-reducing end andcan accommodate approximately, two to threesugar molecules (Figure 7(a)), which correlateswith the exolytic activity of this enzyme. Bindingof a longer carbohydrate would require substantialrearrangement of these two loops and apparentlydoes not occur frequently. The open cleft of theFlavoAC does not impose such a constraint andallows binding to the middle of a long carbo-hydrate (Figure 7(b)).


Materials and Methods

Protein purification

The protein was purified from its natural host atSeikagaku Corporation. To stimulate expression of chon-droitin AC lyase II, A. aurescens was grown under thefollowing culture conditions: medium (32.5 l) containing0.4% (w/v) peptone (Kyokuto Pharmaceutical IndustryCo. Ltd, Tokyo), 0.4% (w/v) Ehrlich’s fish extract(Kyokuto Pharmaceutical Industry Co. Ltd, Tokyo) and0.75% (w/v) chondroitin sulfate C (Seikagaku Co.,Tokyo); initial pH 6.2; aeration rate 1 vessel volume perminute; agitation speed 220 rpm; cultivation time 24hours. The enzyme was purified essentially asdescribed13 but with some modifications. Briefly, afterbacterial cells were pelleted by centrifugation at 15,000gfor 15 minutes, solid ammonium sulfate was added tothe supernatant fluid up to 75% saturation. At 4 8C, 75 gof the protein precipitate (50,000 units) was dialyzedagainst 20 mM sodium acetate (pH 5.2) and loaded ontoan SP-Sepharose (Amersham Bioscience Corp, Piscat-away, NJ) column (2.6 cm £ 70 cm) pre-equilibratedwith the same buffer. The enzyme was eluted with alinear gradient from 20 mM to 300 mM sodium acetatebuffer (pH 5.2). Enzyme activity and protein amountswere monitored as described.13 This chromatographyprocedure was repeated three times and the enzyme-containing fractions collected and concentrated by

ultrafiltration (Ultrafilter Type P0200, cut-off 20,000 Da,Advantec, Tokyo Japan) under N2: Finally, the enzymewas loaded onto a Sephacryl S-200 HR gel-filtrationcolumn (Amersham Bioscience Corp, Piscataway, NJ)equilibrated with 0.01 M sodium acetate buffer (pH 5.6)and eluted with the same buffer. The fractions showingthe highest specific activity and high purity by SDS-PAGE were pooled and used in crystallizationexperiments.

Oligosaccharide preparation

Chondrotin 4-sulfate tetrasaccharide (CStetra) and hya-luronan tetrasaccharide were prepared and characterizedas described.10 Briefly, chondroitin 4-sulfate from bovinetrachea and dermatan sulfate from porcine intestinalmucosa were subjected to controlled depolymerizationusing chondroitin ABC lyase and the reactions were ter-minated prior to completion by boiling for five minutes.Each oligosaccharide mixture was separated on a Bio-Gel P6 column and fractions consisting of tetrasacchar-ides and hexasaccharides were collected. These mixtureswere further fractionated by strong anion-exchangeHPLC, single oligosaccharides were obtained, and theirpurity confirmed by capillary electrophoresis and theirstructures by MS and NMR analyses. The structure ofCStetra was DUAp (1 ! 3)-b-D-GalpNAc4S (1 ! 4)-b-D-GlcAp (1 ! 3)-a,b-D-GalpNAc4S, where DUAp is theunsaturated sugar residue, 4-deoxy-a-L-threo-hex-4-is

Figure 6. Stereo view of the conformation of the 460–469 loop in native and complexed ArthroAC (blue) and inArthroAC-Hg (magenta). The location of the thimerosal Hg atom near the Cys408 in the open conformation is shownas a magenta ball. The side-chains of His233, Arg296, Glu407 and Trp465 are shown explicitly with the hydrogenbonds marked in broken lines. The þ2 and þ1 sugars are also shown.


enopyranosyluronic acid; GlcAp is glucopyranosyl-uronic acid; GalpN is 2-deoxy-2-aminogalactopyranose;S is sulfate; and Ac is acetate.

Protein crystallization and data collection

Needle-shaped crystals of ArthroAC were reportedmany years ago;13 however, they were not characterized.

We obtained crystals by the hanging-drop, vapor-diffu-sion method in drops containing 2 ml of protein (10 mg/ml) and 2 ml of reservoir solution (23% (w/v) PEG8000,0.1 M sodium phosphate buffer (pH 6.4), 0.4 Mammonium acetate, 10% (v/v) glycerol) suspendedover 1 ml of reservoir solution. Small crystals appearedwithin a few days. Larger crystals were obtained bymacroseeding with precipitant concentration lowered to21% (w/v).

Figure 7. Stereo view of the molecular surface within the extended substrate binding site of (a) ArthroAC–tetrasac-charide complex. The insertions in the sequence capping the substrate binding site are shown in green. (b) FlavoAC,with bound tetrasaccharides. The Figure was prepared with the program GRASP.54


The crystals belong to the monoclinic space group P21

with cell dimensions a ¼ 57:6 A, b ¼ 85:5 A, c ¼ 80:5 A,b ¼ 106.98 and contain one molecule in the asymmetricunit. Prior to data collection the crystals were immersedfor ten seconds in a cryoprotectant solution containing22.5% (w/v) PEG8000, 0.1 M sodium phosphate buffer(pH 6.4), 0.4 M ammonium acetate and 20% (v/v)glycerol, mounted in a nylon loop and flash-cooled in acold stream of N2 gas to 100 K. These crystals diffractedto 1.3 A resolution at the X8C beamline at BrookhavenNational Laboratory.

Crystals of the protein complexed with thimerosalwere obtained by an overnight soak of native crystals inthe cryoprotectant solution containing 2 mM Hg salt.These crystals were non-isomorphous with the nativecrystals and had cell dimensions: a ¼ 57:4 A, b ¼ 85:3 A,c ¼ 82:2 A, b ¼ 105.88. Multiwavelength anomalous dif-fraction (MAD) data at three wavelengths were collected(Table 7).

Enzyme–substrate complexes

To obtain complexes of ArthroAC with its substrateswe resorted to a series of soaks of the native crystals incryoprotectant solution containing the substrate fortimes ranging from 30 seconds to ten hours (Table 2).Native crystals were soaked in the cryo-protecting solu-tion containing 5 mM CStetra or HAtetra for a specifiedlength of time, then flash-frozen in a cold N2 gas stream(100 K) on the detector and used immediately for datacollection. There was no significant change in cell dimen-sions as compared to the crystals of native ArthroAC(Table 2).

Diffraction data were collected at the X8C beamline,NSLS, Brookhaven National Laboratory, using theQuantum-4 CCD detector. The highest-resolution data,1.25 A, were obtained from the native crystal soaked forten hours in 5 mM CStetra. Data processing and scalingwas performed with HKL2000.43 Data collection statisticsare shown in Tables 2 and 7.

Structure determination and refinement

The structure of ArthroAC was solved from the Hg-

containing thimerosal derivative of ArthroAC. Analysisof the MAD datasets using the program SOLVE44

revealed three Hg sites in the asymmetric unit. Thesesites were used to calculate experimental phases to aresolution of 1.3 A and resulted in an overall figure ofmerit (FOM) of 0.33–1.3 A. Electron density modificationperformed with the program RESOLVE45 assuming asolvent content of 0.4 led to a significant increase ofthe FOM (0.45 at 1.3 A resolution) and substantiallyimproved the electron density map. Approximately,80% of the protein main chain was built automaticallyusing the program RESOLVE. Additional fragments ofthe main chain and many side-chains were built manu-ally using the program O.46 Since the primary sequenceof the protein was unknown, the amino acid type foreach residue was selected to fit the experimental electrondensity map. At 1.3 A resolution, most of the assign-ments were unambiguous and nearly the entire chainwas traced in the initial map. This initial sequenceassignment was adjusted during the progress of refine-ment as the electron density features improved. Initialrefinement was performed with the program CNS, ver-sion 1.147 and the model was rebuilt manually using theprogram O. Subsequently, refinement was continuedusing the program REFMAC5, version 5.1.08.48 Duringrefinement of this model, 1% of the reflections were setaside for the calculation of Rfree: Water molecules wereinitially added automatically with the program CNSand subsequently updated and corrected by visualinspection of the difference map. The final model of thethimerosal-soaked crystal has been refined to an R-factorof 0.134 and Rfree of 0.156 at 1.3 A resolution (Table 3).The current model contains 754 residues (Pro4–Arg757).The final amino acid sequence of the model was derivedfrom the combination of electron density maps and massspectrometry data.

Refinement of native protein and enzyme–substrate complexes

The model of Hg-bound ArthroAC was taken as astarting point for refinement of the crystal structure ofthe native protein. Refinement was performed with theprogram REFMAC5. For monitoring of Rfree duringrefinement, 1% of reflections were set aside. The electrondensity map showed that one loop had a substantiallydifferent conformation and was rebuilt manually. Thismodel was refined at 1.35 A resolution to a final R-factorof 0.130 and Rfree of 0.175. The model contains residues4–757, 1025 water molecules, one sodium ion and onephosphate ion (present at 0.1 M concentration in themother liquor).

This model, in turn, was used to determine the struc-tures of all the complexes of chondroitin AC lyase withchondroitin 4-sulfate tetrasaccharide (CStetra) with differ-ent soaking times (30 seconds, two minutes, ten minutes,35 minutes, two hours, four hours, ten hours) and of theHAtetra complex (two minutes soaking time). It was clearfrom the height of peaks in the difference electron dens-ity map that the occupancies of sugars in sites (22, 21)and (þ1, þ2) differed systematically from structure tostructure, in a manner consistent with the enzyme beingactive in the crystal. In such a case, the initial concen-tration of tetrasaccharide would decrease with time,while that of a disaccharide product increases. At anytime, active sites of some enzyme molecules mightbe temporarily empty. Previous structural studies onchondroitin lyase showed that disaccharides were found

Table 7. MAD data collection statistics

Hg—peak

Hg—inflection

Hg—remote Native

Wavelength (A) 1.005133 1.009078 0.997068 0.950000a (A) 57.3 57.6b (A) 85.2 86.5c (A) 82.1 80.5b (deg.) 105.8 106.9Resolutionrange (A) (lastshell)

40–1.3(1.35–1.30)

40–1.3(1.35–1.30)

50–1.4(1.45–1.40)

50–1.3(1.35–1.30)

Rsym (last shell) 0.066(0.274)

0.067(0.325)

0.067(0.221)

0.086(0.540)

Completeness(%) (last shell)

98.4(85.0)

98.8(89.0)

99.0(90.7)

96.4(93.4)

I=sðIÞ (lastshell)

14.9(5.3)

14.7 (6.8) 13.7(4.5)

8.6 (3.4)

Total reflections 876,240 884,015 721,975 1,294,307Unique reflec-tions

182,880 183,424 147,941 178,066

Redundancy 4.8 4.8 4.9 7.3


predominantly in subsites (22, 21), and not in (þ1,þ2).10 Therefore, at any given time there is a mixture oftetrasaccharides bound to sites (22, 21, þ1, þ2) anddisaccharides bound predominantly to sites (22, 21).For that reason, the occupancies of sites (22, 21) areexpected to be higher than for sites (þ1, þ2). To obtainan estimate of the relative occupancies at the 2 and þsites, we assumed that, since the substrate is boundtightly to the enzyme through numerous hydrogenbonds, stacking and van der Waals interactions, the tem-perature factors of the substrate are similar to those ofthe residues with which it interacts. We have adjustedindependently the occupancies of sites (22, 21) and(þ1, þ2) in steps of 0.1 until the average temperaturefactor of the substrate was close to that of the surround-ing atoms (Table 4). The derived occupancy values pro-vide information about the relative rather than absoluteoccupancies. In the HAtetra complex, only two sugarrings were visible in the difference electron densitymap, in positions (22, 21) (reaction product). Fullstatistics of refinements are summarized in Table 3.

PROCHECK49 showed that all models have good geo-metry with no outliers.

Protein Data Bank accession numbers

Coordinates of the native chondroitinase AC(ArthroACnat), the mercury derivative (ArthroACHg),the two minutes soaked complex with hyaluronan(ArthroACHA), and 30 seconds, ten minutes and tenhours soaked complexes with CStetra (ArthroACCStetra) aredeposited in the Protein Data Bank, RCSB, with acces-sion codes 1RW9, 1RWA, 1RWC, 1RWF, 1RWG, 1RWH.

Amino acid sequence assignment

As mentioned above, the amino acid sequence of theprotein has not been previously determined. The highresolution of the diffraction data and the parallelindependent refinement of several structures (native, thi-merosal derivative and several enzyme–substrate com-plexes) allowed us to deduce the sequence directly fromthe electron density maps. This sequence was confirmedand corrected in several places by mass spectrometryanalysis. For each of the five best datasets at resolutions1.25–1.5 A, the refinements converged at very low R-factors of 0.114–0.138 (R-free of 0.142–0.178), indicatinghighly refined and reliable structures.

Electron density-based assignments

At 1.3 A resolution the residue types Trp, Tyr, His,Arg, Lys, Pro, Ile, Leu are defined unambiguously bytheir shape (Supplementary Material). The three highestpeaks in the MAD-derived experimental map corre-sponded to Hg atoms and each was associated with acysteine residue. The subsequent 12 highest peaks occurin the middle of the side-chain electron densities andwere associated either with a cysteine or a methionineresidue, based on the shape of the electron density. Thenext group of peaks corresponded to main chain orside-chain oxygen atoms. During the refinement, threeadditional residues on the protein surface were assignedas methionine based on the refined shape of the electrondensity. A total of seven cysteine and 11 methionineresidues were identified. The distinction between Valand Thr could be made based on the height of the elec-tron density for CG2 versus CG1/OG1, often supported

by the presence of hydrogen bond donors or acceptorswithin 3 A. The assignment of Phe was rather straight-forward. The remaining amino acid types, Ser, Ala andGly, were assigned based on the shape of the electrondensity.

The electron density was convincing for Asx and Glx.Initially, all Asx were refined as Asp and all Glx as Glu.When the R-factor dropped below 0.2 we reassignedthese residues to either Gln (Asn) or Glu (Asp) basedon: (1) the height of peaks corresponding to the side-chain atoms; (2) the hydrogen bonding network and thedirectionality of hydrogen bonds; (3) sequence conserva-tion in related protein sequences. All these assignmentswere re-evaluated during each rebuilding cycle and cross-checked between all independently refined structures.

Mass spectrometry-based assignments

Mass spectrometry was applied to determine the con-sistency between the measured molecular mass of theentire molecule and trypsin-derived peptides and thepredicted molecular mass based on the establishedamino acid sequence. The molecular mass of the entireprotein was determined on an LC/MS Agilent 1100mass spectrometer. For MS/MS analysis the protein wasin-gel digested with trypsin. Peptides were separated ona CapLC HPLC (Waters, Milford, USA) with 0.1% (v/v)aqueous formic acid and 0.21% (v/v) formic acid inacetonitrile used for the gradient composition. A volumeof 20 ml of in-gel digest sample was injected onto thetrapping column at a flow-rate of 15 ml/minute andthen washed for five minutes with 0.1% (v/v) aqueousformic acid. The peptides from the trapping columnwere directed to the PicoFrite analytical column (NewObjective, MA) filled with 10 cm of C18 BioBasice pack-ing (5 mm, 300 A, 75 mm ID £ 10 cm). The spraying tipof the PicoFrite column was positioned near thesampling cone of the mass spectrometer and the capil-lary voltage adjusted to achieve the best plume possible.The analysis was done on a QTOF-2 mass spectrometer(Micromass, UK) upgraded with EPCAS electronics. Theinstrument was set in data directed analysis (DDA)mode, where an MS survey scan from 350 to 1600 m/zwas recorded in one second, then the strongest ion wasselected for MS/MS (50–2000 m/z) for duration of onesecond. The DDA was set to select doubly and triplycharged ions. The instrument then switched back to theMS survey to select the second most intense ion for thenext MS/MS spectrum. The mass spectrometric analysiswas designed to collect a maximum of different ions toget a maximum of protein coverage. The interscan timewas set to 0.1 second.

The optimized lyase sequence of 757 amino acid resi-dues when matched with the experimental 317 tandemmass spectra resulted in the assignment of 157 of the317 tandem MS spectra that identified 37 tryptic frag-ments (645 residues) that covered 85.2% of the sequence.Mascot search parameters were restricted to oxidation ofmethionine, histidine and/or tryptophan, alkylation ofcysteine by iodoacetamide or in-gel during electrophor-esis by acrylamide, and the deamidation of asparagine/glutamine or the carbamidomethyl derivative of cys-teine, with a parent ion mass tolerance of 0.5 Da andallowing for one missed cleavage. Relaxing the strin-gency of the search to allow for non-specific proteolysisby trypsin (and maintaining the parameters above)resulted in the identification of a further 28 (non-tryptic)fragments defined by 35 tandem MS spectra with a


concomitant increase in coverage by 25 amino acid resi-dues to 88.5%, while further decreasing the stringencyof the matching by increasing the tolerance on the parentpeptide mass to 2 Da resulted in the assignment ofanother ten spectra to the sequence. In total, 202 of thetandem MS spectra were matched to the best sequencein this manner. The quality of the remaining MS/MSspectra was insufficient for confirmation of sequencealthough sequence tags50 of low confidence could be gen-erated and about 42 of the spectra were weak, sparse andpossibly correspond to background noise. An accountingof the peptides as predicted from the X-ray data but notobserved by tandem MS indicated that several shorttryptic peptides (residue numbers 44–50, 51–57, 175–176, 295–296, 297–300, 342–343, 356–360, 385–388,496–501, 502, 586–588, 639, 640–645, 646–650, 662–664,752–754, 755–756, 757) that account for 64 residueswere most likely lost during the loading/washing of thereversed-phase LC column, whereas the rather largetryptic fragment (residues 503–525) that is well definedby the X-ray data may have remained in the gel duringthe robotic in-gel digestion/extraction and/or remainedbound to the reversed-phase column throughout theLC-QToF MS analysis. Results of Mascot analysis andsamples of MS/MS spectra for several peptides aredeposited as Supplementary Material.

An example of the experimental and the refined elec-tron density map for the region around Asn183 isshown in Supplementary Material. Finally, the sequenceof ArthroAC derived by combining information fromelectron density maps and mass spectrometry wasaligned with related enzymes based on their structuralsuperposition (Figure 1).

At completion of this process we were confident of theidentity of 746 out of 754 modeled residues (98.9%). Forthree additional residues (Asn/Asp and Gln/Glu) weare less certain of the assignment and it is based primar-ily on differences in the B-factors and potential formationof hydrogen bonds. Finally, five residues located in flex-ible loops exposed to solvent and with B-factors of morethan 30 A2 (average B for the protein is ,15 A2) and forwhich no fragment was found in MS/MS data couldnot be identified unambiguously. These residues are dis-tant from the substrate binding site (marked in Figure 1).

At the end of the structure determination process wehave determined the N-terminal amino acid sequence of21 residues of a dissolved crystal by Edman degradation.The first three N-terminal residues are disordered in thestructure, the identity of the subsequent 18 residuesagrees with the electron-density-based assignments.

Kinetic analysis

Glycosaminoglycans CS-A (19,000 Da), CS-C (43,000 Da),CS-D (30,000 Da), low molecular mass hyaluronic acid(50,000 Da) and hyaluronic acid (1,000,000 Da) wereobtained from Seikagaku Corporation (Tokyo, Japan).

Arthro AC was dissolved at concentration of 50 mU/mlin 0.1% (w/v) BSA (solution A). Each chondroitin sulfateand lower molecular mass of hyaluronic acid was dis-solved in distilled water at concentration of 10 mg/ml,whereas hyaluronic acid was dissolved in it at concen-tration of 2 mg/ml, respectively. From 10 ml to 200 ml ofsolution containing each GAG thus prepared was addedto 160 ml of 0.4 M sodium acetate buffer (pH 6.0) andthe appropriate amount of distilled water was added toeach solution to adjust the final volume to 700 ml (solu-tion B). Each solution B was incubated at 37 8C. After 30

seconds, 100 ml of solution A was added to solution Band the increased absorbance (ABS) at 232 nm due tothe generation of unsaturated bonds in disaccharidescaused by Arthro AC was monitored at every 30 secondsby spectrophotometer (UV 160A, Shimadzu, Kyoto,Japan). Reaction velocities (v) at different concentrationof the GAGs were plotted as DABS=Dt: Since all GAGsused in the experiment were catalyzed to disaccharideunits, we calculated substrate concentrations of theGAGs as molar basis of disaccharide units contained ineach GAG. For example, in the case of the CS-A, it wascomposed of 95.5% of mono-sulfated disaccharide unit(503.3 Da), 3.0% of di-sulfated disaccharide unit (605.3 Da)and 1.6% of non-sulfated saccharide unit (401.3 Da).Therefore, mean molecular mass of the CS-A as disac-charide unit can be calculated to be 505.2. Accordingly,mean molecular mass of the CS-C, the CS-D and bothhyaluronic acid from the disaccharide-unit basis werecalculated as 511.2 Da, 532.2 Da and 401.3 Da, respec-tively. By use of substrate concentration based on thiscalculation, a Lineweaver–Burke plot was obtained, andV and KM of every GAG were determined.

Acknowledgements

We thank Drs Allan Matte, Joseph D. Schrag andJ. Sivaraman for help with data collection,Ms France Dumas for amino acid sequencing,Ms Christine Munger, Dr Daniel Boismenu,Mr Sajid Karsan and Montreal Proteomics Networkfor mass spectrometry analysis. This work wassupported, in part, by CIHR grant 200009MOP-84373-M-CFAA-26164 to M.C. and NIH grantsGM38060 and HL62244 to R.J.L.

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Edited by I. Wilson

(Received 23 September 2003; received in revised form19 December 2003; accepted 29 December 2003)

Supplementary Material comprising Figuresshowing electron density maps at various contourlevels and Tables and plots displaying peptideidentification by mass spectrometry is available onScience Direct


high-resolution crystal structure of arthrobacter aurescens chondroitin … · high-resolution...

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