Structure and function of a serine carboxypeptidaseadapted for degradation of the proteinsynthesis antibiotic microcin C7Vinayak Agarwala,b, Anton Tikhonovc,d, Anastasia Metlitskayac, Konstantin Severinovc,d,e, and Satish K. Naira,b,f,1

aCenter for Biophysics and Computational Biology, bInstitute for Genomic Biology, and fDepartment of Biochemistry, University of Illinois atUrbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801; cInstitutes of Molecular Genetics and Gene Biology, Russian Academy of Sciences,Moscow 11934, Russia; eDepartment of Molecular Biology and Biochemistry, and dWaksman Institute, Rutgers, State University of New Jersey,Piscataway, NJ 08854

Edited by Perry Allen Frey, University of Wisconsin, Madison, WI, and approved January 20, 2012 (received for review August 30, 2011)

Several classes of naturally occurring antimicrobials exert theirantibiotic activity by specifically targeting aminoacyl-tRNA synthe-tases, validating these enzymes as drug targets. The aspartyl tRNAsynthetase “Trojan horse” inhibitor microcin C7 (McC7) consists of anonhydrolyzable aspartyl-adenylate conjugated to a hexapeptidecarrier that facilitates active import into bacterial cells through anoligopeptide transport system. Subsequent proteolytic processingreleases the toxic compound inside the cell. Producing strainsof McC7 must protect themselves against autotoxicity that may re-sult from premature processing. The mccF gene confers resistanceagainst endogenous and exogenous McC7 by hydrolyzing theamide bond that connects the peptide and nucleotide moieties ofMcC7. We present here crystal structures of MccF, in complex withvarious ligands. The MccF structure is similar to that of dipeptideLD-carboxypeptidase, but with an additional loop proximal to theactive site that serves as the primary determinant for recognitionof adenylated substrates. Wild-type MccF only hydrolyzes thenaturally occurring aspartyl phosphoramidate McC7 and syntheticpeptidyl sulfamoyl adenylates that contain anionic side chains.We show that substitutions of two active site MccF residues resultin a specificity switch toward aromatic aminoacyl–adenylate sub-strates. These results suggest howMccF-like enzymes may be usedto avert various toxic aminoacyl–adenylates that accumulate dur-ing antibiotic biosynthesis or in normal metabolism of the cell.

tRNA synthetase inhibitor ∣ self-immunity ∣ reaction mechanism ∣protein engineering

The influx of genome sequence data has resulted in the iden-tification of various targets in pathogenic species for antibiotic

development. Among promising targets are the 20 aminoacyl-tRNA synthetases (aaRSs) that are essential for protein synthesis.Aminoacyl-tRNA synthetases catalyze the attachment of thecorrect amino acid to cognate tRNA and thus are fundamentalto translation of the genetic code (1, 2). The acylation reactionfirst involves the formation of an enzyme bound aminoacyl–ade-nylate complex, followed by the nucleophilic attack of the 2′ or 3′hydroxyl of the terminal ribose of the tRNA on the adenylate toform the aminoacyl-tRNA and adenosine monophosphate (1).Inhibition of either one of these two enzymatic steps disruptstRNA charging, which would stall elongation of growing polypep-tide chains, followed by limited bacterial growth and attenuationof virulence in vivo (3).

One subset of aaRS inhibitors consists of nonhydrolyzable ana-logs of the aminoacyl–adenylate intermediate that is generatedduring the aaRS reaction. However, synthetic aminoacyl–adeny-lates analogs are poor drug candidates because (i) they inhibithuman aaRSs, and (ii) most lack in vivo activity due to imper-meability into bacterial cells (4). Nature has overcome both ofthese limitations by employing a Trojan horse strategy, in whichthe drug molecule is conjugated with a specialized module thatfacilitates active transport into bacterial cells where antibiotic

activity is exerted after intracellular processing. Examples ofnaturally occurring antibiotics that employ this Trojan horse strat-egy include the LeuRS inhibitor agrocin 84 (in which the toxicgroup is linked through a phosphoramidate bond to a D-glucofur-anosyloxyphosphoryl moiety) (5), the SerRS inhibitor albomycin(a toxic group covalently linked to a hydroxymate siderophore)(6), and the AspRS inhibitor microcin C7 (Fig. 1A, 1) (McC7;consisting of a modified aspartyl-adenylate linked to a six-residuepeptide carrier).

Following active transport into target cells through the YejA-BEF transporter, the McC7 heptapeptide precursor is processedby nonspecific cellular aminopeptidases to release the toxic com-pound, processed McC7 (7) (Fig. 1A, 2). Producing organismsmust protect themselves against the premature, unwanted proces-sing that can result in toxicity. In the case of agrocin 84 andalbomycin, the antibiotic biosynthesis clusters encode additionaltRNA synthetases (LeuRS in the case of agrocin 84, SerRS in thecase of albomycin), which are resistant to inhibitors. The McC7biosynthetic cluster does not encode a tRNA synthetase but in-stead, employs specialized enzymes that act on processed and/orintact McC7 in the producing cells. One such autoimmunitydeterminant is encoded by themccF gene, whose product proteo-lytically hydrolyzes the amide bond connecting the terminalaspartate and modified AMP in McC7 (8). MccF was shown toinactivate both intact and processed forms of McC7. The enzymefunctions only on substrates that bear an acidic aspartyl (aspartylsulfamoyl adenylate, DSA) (Fig. 1A, 3) or glutamyl sulfamoyladenylate (ESA) (Fig. 1A, 4) moiety conjugated to adenosine,with diminished activity also observed for seryl conjugatedadenosine (8).

In order to elucidate determinants of substrate recognition,we have determined the high-resolution crystal structure of wild-type MccF and of a catalytically inactive variant in complex withnatural and synthetic substrates. Analysis of these structures re-veals how the repertoire of substrates accepted by serine carbox-ypeptidases can be expanded to include nucleotide-containingpeptide antibiotics through π-stacking interactions. The crystalstructures, along with kinetic characterization of structure-guidedmutants, provide a firm structural framework for understandingthe substrate recognition, and catalysis by MccF, which are

Author contributions: V.A., K.S., and S.K.N. designed research; V.A. performed research;A.T. and A.M. contributed new reagents/analytic tools; V.A. and S.K.N. analyzed data;and V.A., K.S., and S.K.N. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have been deposited inthe Protein Data Bank, www.pdb.org (PDB ID codes: MccF wild type, 3TLA; MccF S118AApo, 3TLG; MccF S118A McC7, 3TLC; MccF S118A DSA, 3TLB; MccF S118A ESA, 3TLE).1To whom correspondence should be addressed. E-mail: s-nair@life.illinois.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1114224109/-/DCSupplemental.

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further borne out by our engineering experiments that yield anenzyme with a drastically altered substrate scope.

ResultsIn Vitro Kinetics of MccF-Catalyzed Reaction. We had previously de-monstrated that overexpression of themccF gene results in McC7

resistance, and that recombinant MccF cleaved the amide bondthat connects the peptidyl and nucleotidyl moieties of McC7 (8).Similar effects were also observed with the synthetic DSA andESA (Fig. 1B). We further investigated the MccF-catalyzed reac-tion by determining steady-state kinetic parameters.

In order to determine the kinetic parameters for ESA hydro-lysis reaction, we utilized a continuous coupled assay that mon-itored the formation of glutamate upon hydrolysis of ESA.Kinetic measurements for the hydrolysis of DSA were performedby direct detection of reaction product (sulfamoyl adenosine)using analytical HPLC. Steady-state kinetic parameters for thehydrolysis of processed McC7 could not be determined usingeither of these methods. However, as the mode of binding of pro-cessed McC7 is essentially identical to that of DSA (see below),we assume that the kinetic parameters for processed McC7 hy-drolysis would be very close to those for DSA. The catalytic effi-ciency (kcat∕Km) for the hydrolysis of DSA was 9.37E4 M−1 s−1,and is approximately eightfold greater than that for ESA(1.13E4 M−1 s−1) (Fig. 1C and Table 1). Mutation of the activesite catalytic serine residue to alanine rendered the enzyme com-pletely devoid of measurable catalytic activity. Notably, for thewild-type enzyme, no hydrolysis could be observed with sulfamoyladenylates that contained aromatic amino acids, such as pheny-lalanyl sulfamoyl adenylate (FSA) (Fig. 1A, 5).

MccF is aMember of the Serine LD-Carboxypeptidase Superfamily.Thestructure of wild-type MccF has been determined to 1.2-Å reso-lution by single wavelength anomalous dispersion data collectedfrom crystals of selenomethionine-labeled protein (relevant datacollection statistics are given in Table S1). The structure of MccFcan be divided into two distinct domains: an amino terminal re-gion (residues Pro7–Thr164) consisting of four parallel β-sheetsflanked by α-helices, and a carboxy-terminal region (Lys209–Ser341) composed of a seven-strand β-sandwich with three α-he-lices situated along one face of the strands. The two domains arelinked through an unusually long loop (Arg165–Gly208), whichwe term the “catalytic loop” (Fig. 2A). Numerous contacts withthe side- and main-chain atoms of both domains fix the positionof the catalytic loop. Consistent with the behavior of MccF insolution as a dimer, the crystallographic asymmetric unit consistsof an MccF homodimer (Fig. 2B) and a total surface area of over3;681 Å2 is occluded upon dimerization. The overall topology ofMccF is similar to LD-carboxypeptidases that cleave the linkagebetween meso-diaminopimelic acid and D-alanine during pepti-doglycan recycling (Protein Data Bank ID code 1ZRS; Z score14.2; rmsd of 1.9 Å over 274 aligned Cα atoms) (9). However,the catalytic loop is absent from LD-carboxypeptidases and istherefore unique to MccF (Fig. S1).

Fig. 1. Chemical structures of microcin C7 and analogs and demonstrationof MccF hydrolysis activity. (A) Chemical structures of microcin C7 (1) and itsprocessed form (2), along with synthetic sulfamoyl adenylates of aspartate(3), glutamate (4), and phenylalanine (5). The site of hydrolysis by wild-typeMccF is marked by an arrow. (B) Hydrolysis of DSA by wild-type MccF enzymedemonstrated by HPLC separation the substrate and reaction product, sulfa-moyl adenosine. (C) Michaelis–Menten kinetic curves for the hydrolysis ofDSA (⦁) and ESA (○) by wild-type MccF enzyme.

Table 1. Steady-state kinetic parameters for DSA and ESAhydrolysis by MccF wild-type and mutant enzymes

Km, μM kcat, s−1kcat∕Km,M−1 s−1

Relativekcat∕Km*

DSAWild type 74.31 6.96 9.37E4 1.000N220A 186.32 2.22 1.19E4 0.126K247A 133.92 3.27 2.44E4 0.260S118A ND ND

ESAWild type 51.64 0.58 1.13E4 1.000W186F 218.77 0.39 1.78E3 0.157W186A ND NDR246A 154.27 0.56 3.64E3 0.322N220A 105.89 0.62 5.85E3 0.518K247A 238.92 0.08 3.51E2 0.031N220A/K247A ND ND

ND, no detectable activity precluded kinetic parameter determination.*The kcat∕Km of mutant enzyme relative to wild-type enzyme.

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A catalytic triad, composed of Ser118, His311, and Glu243,is situated at the interface between the two domains (Fig. 2A),consistent with the earlier report that mutation at Ser118 resultsin a loss of catalytic activity (8). The side chain of Glu243 is buriedin a polar environment with one of the carboxylate oxygen atomshydrogen bonded to the Nδ1 of His311 (2.9 Å) and the secondcarboxylate oxygen atom hydrogen bonded to the Oη of Tyr137(2.5 Å) and to Nδ2 of Asn218 (3.0 Å). The Nϵ2 of His311 isappropriately placed at a distance of 2.8 Å from the Ser118side-chain hydroxyl where it can abstract the proton to generatethe catalytic alkoxide anion for nucleophilic attack at the scissilepeptide bond (10). Rather unexpectedly, the Ser118 side chainexists in two different conformations and a hydrogen-bondinginteraction with His311 is possible in only one of these confor-mations.

We also determined the 1.5-Å resolution crystal structure ofthe MccF Ser118Ala mutant and found it to be nearly identicalto the wild-type enzyme structure (Table S1). Thus, the loss ofcatalytic activity for the mutant is due to the loss of the catalyticserine side-chain hydroxyl nucleophile. Subsequent discussionsof the substrate cocrystal complexes of the Ser118Ala MccF aredescriptive of the productive state for substrate binding to thewild-type enzyme.

The Catalytic Loop Contributes Directly to Substrate Engagement. Inorder to delineate the molecular basis for substrate recognition,we determined the 1.3-Å resolution cocrystal structure of Ser118-Ala MccF in complex with processed McC7. Difference Fourieranalysis reveals unambiguous electron density correspondingto processed McC7 in the vicinity of the active site (Fig. 3A). Fol-lowing the Schechter and Berger nomenclature (11) for serineproteases, we refer to the nucleotide binding site as the P1 siteand the aspartyl side chain of processed McC7 occupies the P1′site. The interactions at the P1 site are primarily modulated byπ-stacking interactions of the adenine ring of McC7 againstthe indole side chain of Trp186, located within the catalytic loop.The 2′ and 3′ hydroxyl groups of the ribose sugar are respectivelypositioned 2.9- and 2.7-Å away from the side-chain carboxylateoxygen atoms of Glu277 and engage in hydrogen-bonding inter-actions (Fig. 3A). The 2′ hydroxyl is also positioned 2.7-Å awayfrom guanidine side chain of Arg246, which in turn is also hydro-gen bonded to Ser183 side-chain hydroxyl. At the P1′ site, one ofthe carboxylate oxygen atoms of processed McC7 aspartate ispositioned 3.1-Å away from the Nζ of Lys247 and the other oxy-gen atom is 2.9-Å away from Nδ2 of Asn220. This carboxylateoxygen atom is also engaged in an interaction with the Oγ1 ofThr221 (3.2 Å) and the guanidino side chain of an alternateconformation of Arg254 from the other MccF monomer (N-Odistances of 3.1 and 2.9 Å).

Although π-stacking interactions can provide extensive surfacearea for van der Waals contact, such interactions result in electro-static repulsion as the delocalized π-electrons result in a con-centration of negative electrostatic potential. However, the elec-trostatic potential of heterocyclic adenine and guanine nucleobases

of DNA/RNA differ markedly from that of homocyclic conjugatedsystems such as benzene. Calculations of the electrostatic potentialmap of adenine illustrates that the negative charge is concentratedon N1, N3, and N7, whereas C8 and N9 have significant positivepotential (12). Similar studies on the electrostatic potential oftryptophan suggest a relatively greater negative potential on thesix-member ring of the indole side chain than on the five-memberheterocycle (13). In the MccF cocrystal structures, the side chainof Trp186 and the adenine ring of the substrate are oriented tominimize unfavorable electrostatic interactions between the twoheterocyclic π-electron systems (Fig. S2).

The phosphoramidate that links the adenine and the aspartateof McC7 also interacts with the peptide backbone atoms of theenzyme. The carbonyl oxygen atom of the amide bond of McC7 ispositioned 3.1- and 2.8-Å away from the backbone amide nitro-gen atoms of Asp119 and Gly92 residues, respectively. One of thephosphate oxygen atom is positioned 2.9-Å away from the Gly91amide nitrogen. These interactions would stabilize the oxyanionof the tetrahedral acyl-enzyme intermediate formed upon attackof the Ser118 hydroxyl at the carbonyl (10). No other amino acidside chains are positioned to favorably interact with the oxygenatom. Unexpectedly, the propylamine appendage to the phos-phate that increases the biological activity of McC7 (14) is fullyextended and points outward from the active site, where there areno interactions with the terminal primary amine.

Because we could not determine the kinetic parameters forMcC7 hydrolysis by MccF due to both acid lability of the phos-phoramidate and inability to achieve sufficient HPLC separationof reaction products from unreacted substrate, we utilized DSAas a mimic for processed McC7. In order to confirm that the pro-tein-ligand interactions with DSA are similar to those observedfor processed McC7, we first determined the 1.5-Å resolutioncocrystal structure of inactive Ser118Ala MccF in complex withDSA (Fig. 3B). Briefly, the interactions at the P1 site of the sul-fate oxygen atoms and the stabilization of the oxyanion transitionstate are essentially identical to those observed in the processedMcC7 cocrystal structure. The carboxylate oxygen atoms of theaspartate side chain at P1′ are also similarly engaged by Asn220and Lys247 (Fig. 3B). The similarities in the two cocrystal struc-tures validate the use of DSA as a substrate mimic of processedMcC7 for kinetic analyses.

As noted above, MccF can also utilize ESA as a substrate,albeit with approximately eightfold decrease in catalytic effi-ciency compared to DSA. This substrate preference is reflectedby a turnover number for DSA that is an order of magnitude high-er, whereas the Km for the two substrates is comparable. In orderto further examine the basis for difference in catalytic efficiencywith the two substrates, we determined the 1.3-Å resolution co-crystal structure of Ser118Ala MccF in complex with ESA(Fig. 3C). Although the interactions at the P1 site are essentiallyidentical to those in cocrystal structures with processed McC7and DSA, the interactions with the glutamate side chain at P1′are distinct. Notably, the glutamate side chain is tilted towardLys247 (Oϵ1 and Oϵ2 to Nζ distances of 3.2 and 2.8 Å, respec-

Fig. 2. Overall three-dimensional crystal structureof MccF. (A) Structure of MccF monomer showingthe three domains: amino terminal domain (blue),carboxy-terminal domain (pink), and catalytic loop(green). (B) Structure of the MccF homodimer show-ing the relative positions of the three domains withone monomer colored as above and the secondmonomer colored gray.

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tively), with an additional interaction between Oϵ2 with Nδ2 ofAsn220 (2.6 Å). As a consequence, MccF engages the larger ESAside chain with fewer interactions than either processed McC7or DSA.

Structure-Function Analysis of Site-Specific MccF Mutants. In order toinvestigate the role of active site residues of MccF, we generatedseveral single point substitutions at residues that are engaged ininteractions with the substrate in the cocrystal structures de-scribed above. As the interactions at the P1 site are identical forESA and DSA, ESA was used for kinetic measurements for theMccF P1 site mutant enzymes. In the structures, the catalytic loopresidue Trp186 is the primary determinant for substrate recogni-tion at the P1 site, where it engages in π-stacking interactions withthe substrate adenine ring. Mutation of Trp186 to Phe results ina fourfold increase in the Km for ESA hydrolysis (Table 1). The1.50-Å resolution cocrystal structure of Trp186Phe MccF in com-plex with adenosine monophosphate reveals that the Phe sidechain also engages the adenine ring through π-stacking interac-tions (Fig. S3). The lower substrate affinity of the Trp186Phe mu-tant must be caused by decreased π-stacking interaction. Notably,mutation of Trp186 to Ala resulted in a complete loss of activity,likely as a result of the abolition of the π-stacking interactionswith the substrate nucleotide. The 2′ hydroxyl of the substratenucleotide interacts with Arg246. Substitution of this residue toAla did not alter the kcat of the enzyme, but instead led to a five-fold increase in the Km for the substrate.

At the P1′ site, the carboxylate side chains of the substratesinteract with Lys247 and Asn220. Replacement of Lys247 withalanine led to over 30-fold reduction in the catalytic efficiencytoward ESA. The substitution of Asn220 to alanine resulted inonly a twofold increase inKm and no change in kcat for ESA, lead-ing to twofold decrease in the catalytic efficiency of the enzymefor ESA hydrolysis. The effect of this mutation on kcat∕Km isinsignificant and can be explained by compensatory interactions

of both substrate oxygen atoms of ESA with Lys247 in the mutantprotein. This observation is further exemplified by a much largerdecrease in kcat for DSA hydrolysis for the Asn220 to alanine mu-tation (and an eightfold reduction in the catalytic efficiency) anda lesser decrease in kcat for the Lys247 to alanine mutation. Thetwo carboxylate oxygen atoms of ESA are skewed toward theLys247 side chain, but are nearly equidistant to the Asn220 andLys247 side chains for DSA. Hence the Asn220 to alanine muta-tion cannot be compensated by binding of both carboxylate oxy-gen atoms to Lys247 side chain for DSA. However, substitution ofboth Asn220 and Lys247 to alanine led to complete loss of activityfor both DSA and ESA. Crystal structure of the Ser118Ala/As-n220Ala/Lys247Ala triple mutant enzyme in the apo form con-firmed that this loss in activity is not due to rearrangements atthe P1 site, but rather due to loss in interactions at the P1′ site(Fig. S4).

Alteration of MccF Substrate Scope Through Active Site Mutations.Asshown by our combined biochemical and structural biologicaldata, the stringent specificity of MccF for acidic side chains is dic-tated mainly by the presence of two residues, Asn220 and Lys247,which contact the carboxylate oxygen atoms of processed McC7and its analogues. We hypothesized that alterations at either orboth of these residues could result in a change in substrate scopeof MccF toward substrates that contain aromatic residues. Wegenerated four single mutants and four combinations of doublemutants in which each or both of these residues were mutated toeither leucine or phenylalanine to test the activity of resultantvariants for hydrolysis of FSA. With the exception of Asn220-Leu/Lys247Leu, the mutant proteins were either insoluble orprone to aggregation as judged by analytical size exclusion chro-matography. We carried out time-resolved HPLC analysis ofAsn220Leu/Lys247Leu MccF-catalyzed reaction with FSA as asubstrate and observed that this mutant could indeed hydrolyzeFSA, albeit at a rate much slower than that observed with the

Fig. 3. Stereoviews of MccF-S118A active site withsubstrates bound. (A) Stereoview showing the active sitefeatures of the MccF-S118A in complex with processedMcC7. The MccF carbon atoms are shown in yellowball-and-stick and the McC7 carbon atoms are coloredin green. Superimposed is a difference Fourier electrondensity map (contoured at 2.7σ over background in blue)calculated with coefficients jFobsj − jFcalc j and phasesfrom the final refined model with the coordinates ofMcC7 deleted prior to one round of refinement. (B)Stereoview showing the active site features of theMccF-S118A in complex with DSA. Superimposed is a dif-ference Fourier electron density map (contoured at 2.5σ)calculated as above. (C) Stereoview showing the activesite features of the MccF-S118A in complex with ESA.Superimposed is a difference Fourier electron densitymap (contoured at 2.5σ over background) calculatedas above.

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wild-type enzyme and DSA (Fig. 4 A and B). Electrospray ioniza-tion mass spectrometry analysis confirmed that the site of clea-vage of FSA was at the sulfamoyl amide bond (Fig. S5). Notably,the rate of FSA cleavage by either wild-type MccF or a solubledouble alanine mutant Asn220Ala/Lys247Ala, which were usedas controls, was negligible under similar experimental conditions,demonstrating that hydrophobic aminoacyl-sulfamoyl adenosinesare only accepted as substrates when hydrophobic side chains areintroduced at the P1′ recognition site of the enzyme.

DiscussionMcC7 is a Trojan horse antibiotic that, following processing, mi-mics an aminoacyl–adenylate intermediate to target and inhibitaspartyl tRNA synthetase. Because unwanted, premature proces-sing of McC7 within the producer cells could result in toxicity, theproducing organisms utilize the MccF immunity determinant tohydrolyze the amide bond that connects the peptide and nucleo-tide moieties of McC7. In this study, we present biochemical,kinetic, and structural biological characterization of the hydroly-sis of processed McC7 and its synthetic analogs by MccF. Threeindependently functional domains can be identified in the struc-ture of MccF: The catalytic nucleophile and residues necessaryfor the stabilization of the oxyanion intermediate are providedby the amino terminal domain, and the carboxy-terminal domainprovides residues necessary for catalysis and recognition of theacidic side chain of the substrate.

The principal determinant for substrate binding is provided byTrp186, located in a unique catalytic loop, which stacks with thenucleotide adenine ring of McC7. This catalytic loop is absentin the structurally homologous LD-carboxypeptidases. The lack ofa cocrystal structure of the LD-carboxypeptidase with bound sub-strate precludes a comparison of the substrate binding sites of thetwo enzymes, though the absence of the catalytic loop provides a

possible explanation for the reported inability of LD-carboxypep-tidase to detoxify McC and its analogs (8, 9). We propose that thepresence of the catalytic loop can be used to distinguish proteinswith MccF-like activity from LD-carboxypeptidases that havebeen misannotated in sequence databases (Fig. S1). Analysis ofMccF homologs’ catalytic loop sequences reveals that hydropho-bic residues corresponding to Trp186 are conserved, suggestingthat these proteins should also interact with adenylated targets.Curiously, many MccF homologs are encoded by genes that arenot part of recognizable McC7-like biosynthetic operons. The in-tracellular function of these proteins remains to be determined.

MccF has nearly 10-fold greater turnover number for thehydrolysis of aspartyl sulfamoyl adenylate as compared to that ofglutamyl sulfamoyl adenylate but the Km values for the two sub-strates are comparable. This reflection for substrate preferencethrough a change in kcat and not in Km is reminiscent of earlierwork with serine proteases in which synthetic substrates designedwith altered binding interactions at the P1′ and subsequent down-stream binding subsites have led to nonsignificant changes in theKm for these enzymes, while displaying large variations in the kcat(15–18). For MccF, the order of magnitude increase in kcat forDSA as compared to ESA can be attributed to the differentialmode of stabilization of the acyl-enzyme transition state (formedafter the departure of the adenine sulfonamide) in the active site.Because the aspartate chain makes more contacts with the en-zyme than the glutamate side chain, the activation energy for theformation of this transition state would be lower, leading to anincrease in the rate of hydrolysis of DSA. It should also be notedthat substitutions at the P1 site, Trp186Phe and Arg246Ala, resultin much larger alterations in the Km values for ESA hydrolysisand much less significant changes in kcat values.

An assessment of the rate enhancement for adenylated sub-strate hydrolysis by MccF requires knowledge of the uncatalyzedrate for the cleavage of an amide with a phosphoramide leavinggroup. Although, to our knowledge, such rates have yet to bedetermined, limits for the rate can be estimated to be betweenthe uncatalyzed rate for the hydrolysis of an SD-peptide(2 × 10−10 s−1) (19) and the rate for hydrolysis of a methyl phos-phate dianion (2 × 10−20 s−1) (20). Hence, the rate enhancement(kcat∕knon-cat) for the MccF hydrolysis of DSA can be estimatedto be between 3.5 × 1010- and 3.5 × 1020-fold. This enhancementis comparable to that for the hydrolysis of peptide substrates byserine proteases, such as trypsin and chymotrypsin, that demon-strate roughly 1011-fold enhancement over the uncatalyzed reac-tion rate (10).

The cocrystal structure of MccF in complex with processedMcC7 presents a mechanistic challenge for the protonation ofthe α-amine of the departing carboxy terminus following cleavageof the scissile peptide bond. In serine proteases, the pKa of thecatalytic triad histidine side has been determined to be close to7.0 (21) and consistent with its role in protonating the departingα-NH2 of the peptide, which would have a pKa of greater than9.0. However, in the case of a phosphoramide leaving group ofMcC7, the pKa of the α-NH2 would be close to 4.5 (22), makingprotonation by the catalytic histidine side chain (His311) difficult.The McC7 α-NH2 can only be protonated by either a glutamateor aspartate (pKa lower than 4.5), and no candidate carboxylate ispresent in the vicinity of the departing amine. However, the lonepair of electrons on the α-amine of adenosine phosphoramine arein conjugation with the phosphate oxygen atom, which in turn, iswithin hydrogen binding distance with the backbone amide nitro-gen of Gly91 (Fig. 5). Hence, we propose that, in substrates ofMccF, the α-amine does not need to be protonated to be a leavinggroup, because the developing negative charge on the α-aminenitrogen can then be delocalized by resonance conjugation to thesignificantly more electronegative phosphate oxygen atom.

Fig. 4. Cleavage of hydrophobic sulfamoyl adenosine by mutant forms ofthe MccF enzyme. (A) Time-dependent cleavage of substrate FSA (Inset) byMccF-N220L/K247L mutant enzyme demonstrated by separation of substrateand product peaks by HPLC. (B) Percentage hydrolysis of FSA plotted againstreaction time for MccF-N220L/K247L enzyme.

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Experimental ProceduresProtein Expression, Crystallization, and Structure Determination. Theexpression and purification of recombinant MccF from Escheri-chia coli has been described previously (8). For crystallization,an additional size exclusion chromatography (Superdex 75 16/60;GE Healthcare) was added at the end of purification. MccF wild-type enzyme structure was solved using single wavelength anom-alous diffraction dataset collected on selenomethionine deriva-tized protein crystals and was subsequently used as a searchmodel for structure determination of the substrate cocrystalstructures and structures of the mutant enzymes. Detailed infor-mation is provided in the SI Materials and Methods. Relevant datacollection and refinement statistics are provided in Table S1.

MccF Enzyme Kinetics. Kinetics for the hydrolysis of ESA by MccFwas monitored by a continuous coupled assay to detect the for-mation of glutamate. Kinetics for the hydrolysis of DSA and FSAwere determined by discontinuous separation of the reactantand products by HPLC. Detailed assay and HPLC conditions areprovided in the SI Materials and Methods.

ACKNOWLEDGMENTS. The authors are grateful to Drs. Keith Brister, JosephBrunzelle, and the staff at Life Sciences Collaborative Access Team (ArgonneNational Laboratory) for facilitating data collection. We thank GastonVondenhoff and Arthur Van Aerschot (Rega Institute, Belgium) for providingus with aminoacyl-sulfamoyl adenylates used in this work. This work wassupported, in part, by a Dynasty Foundation Fellowship to A.T., a RussianFoundation for Basic Research Grant to A.M., and a Rutgers UniversityTechnology Commercialization Fund Grant and Russian Academy of SciencesMolecular and Cellular Biology Program Grant to K.S.

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peptidase. J Biol Chem 285:37944–37952.9. KorzaHJ, BochtlerM (2005) Pseudomonas aeruginosa LD-carboxypeptidase, a serine pep-

tidase with a Ser-His-Glu triad and a nucleophilic elbow. J Biol Chem 280:40802–40812.10. Hedstrom L (2002) Serine protease mechanism and specificity. Chem Rev 102:

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glycoside antibiotic kinase APH(3′)-Illa and its nucleotide analogs. Chem Biol9:1209–1217.

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16. Baumann WK, Bizzozero SA, Dutler H (1973) Kinetic investigation of the alpha-chymotrypsin-catalyzed hydrolysis of peptide substrates. The relationship betweenpeptide-structure N-terminal to the cleaved bond and reactivity. Eur J Biochem39:381–391.

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20. Lad C, Williams NH, Wolfenden R (2003) The rate of hydrolysis of phosphomonoesterdianions and the exceptional catalytic proficiencies of protein and inositol phospha-tases. Proc Natl Acad Sci USA 100:5607–5610.

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Fig. 5. Putative mechanism for McC7 hydrolysis by MccF. Hydrogen bond interactions are shown as dashes. MccF catalytic triad residue side-chain atoms arecolored blue, the scissile peptide bond atoms are colored green, and the atoms of the phosphomoiety of the phosphoramidate bond are colored red.

4430 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1114224109 Agarwal et al.

Supporting InformationAgarwal et al. 10.1073/pnas.1114224109SI Materials and MethodsCrystallization of MccF and Ligand Complexes. Crystallization ofthe apo MccF and all variants was carried out using the hangingdrop vapor diffusion technique. Typically, 1 μL of the protein(9 mg∕mL in 20 mM Hepes, 100 mM KCl, pH 7.5) was added to1 μL of precipitant (either 12% PEG 20,000, 0.1 M MES, pH 6.5or 10% PEG 8,000, 8% ethylene glycol, 0.1 MHepes, pH 7.5) andequilibrated against the same solution at 9 °C. Selenomethionine-incorporated MccF was crystallized under similar conditions.Complex with microcin C7 (McC7), aspartyl sulfamoyl adenylate(DSA), or glutamyl sulfamoyl adenylate (ESA) were generated bysoaking apo crystals with 5 mM final concentration of substratecompounds added to the precipitant for 12 h. Crystals were vitri-fied by soaking in precipitant supplemented with 30% ethyleneglycol prior to direct immersion in liquid nitrogen.

X-Ray Crystallographic Data Collection and Structure Determination.Initial crystallographic phases were determined solved by singlewavelength anomalous diffraction utilizing anomalous scatteringfrom the five selenium-substituted methionine residues permonomer of MccF. A fourfold redundant dataset was collectedfrom crystals of selenomethionine-incorporated MccF at the se-lenium absorption edge, to a limiting resolution of 1.45 Å (overallRmerge ¼ 7.2, I∕σðIÞ ¼ 13.7 in the highest resolution shell) utiliz-ing a Mar 300 CCD detector (Life Sciences Collaborative AccessTeam, Sector 21 ID-D, Advanced Photon Source). Data wereindexed and scaled using the HKL2000 package (1). Seleniumsites were identified and refined for phase calculation usingHySS (2), yielding an initial figure of merit of 0.439 (acentric∕centric ¼ 0.454∕0.137) to 1.45-Å resolution. The resultant elec-tron density map was of exceptional quality and permitted mostof the main-chain and 50% of side-chain residues to be automa-tically built using phenix autobuild (3). The remainder of themodel was fitted using Coot (4) and further improved by roundsof refinement with REFMAC5 (5) and manual building. Subse-quent rounds of model-building and crystallographic refinementutilized data from a native crystal of MccF grown under similarconditions that diffracted to 1.30-Å resolution (overall Rmerge ¼6.5, I∕σðIÞ ¼ 6.7 in the highest resolution shell). Cross-valida-tion, using 5% of the data for the calculation of the free R factor,was utilized throughout the model-building process in order tomonitor building bias (6).

The crystal structures of MccF mutants S118A, N220A/K247A/S118A, and W186F in complex with AMP were deter-mined to resolutions of 1.5, 1.7, and 1.5 Å, respectively, andMccF-S118A cocrystal structures with DSA, ESA, and processedMcC7 to resolutions of 1.5, 1.3, and 1.3 Å, respectively, weredetermined by molecular replacement using the coordinates ofnative MccF as a search probe. Each of the structures was refinedand validated using the procedures detailed above. For each ofthe structures, the stereochemistry of the model was monitoredthroughout the course of refinement using PROCHECK (7).

MccF Enzyme Kinetics. Kinetics for cleavage of ESA by MccF wasmonitored by coupling the glutamate production to reducedNADH generation by bovine glutamate dehydrogenase (bGDH)(G2626-50MG; Sigma), which was monitored by continuous in-crease in absorbance at 340 nm using a Cary4000 UV-visible spec-trophotometer. The assays were performed at room temperaturein 20 mM Hepes pH 7.5, 100 mM KCl buffer. NADþ and bGDHconcentrations were kept fixed at 2 mM and 1.7 μM, respectively,in the assays. Linear correlation between the initial rate of reac-

tion and MccF enzyme concentration, at 500 μM ESA substrateconcentration confirmed that the coupling enzymatic reactionwas not rate limiting. MccF wild-type enzyme and W186F,R246A, N220A, and K247A mutants were assayed at 150 nM en-zyme concentration. S118A,W186A, and N220A/K247Amutantswere assayed at 750 nM enzyme concentration. Baseline absor-bance was observed for 1 min before the reactions were initiatedby the addition of enzyme.

Kinetics for the cleavage of DSA by MccF was determined bydiscontinuous HPLC separation of the reactant DSA and productsulfamoyl adenosine (SA). MccF wild-type enzyme and N220Amutant were assayed at 50 nM enzyme concentration. Seventy-five microliter enzymatic reactions were quenched by the addi-tion of equal volumes of 4% formic acid and heating at 80 °Cfor 2 min. HPLC separation was performed on an analytical scaleC18 column (Vydac; 5 μm particle size, 4.6 × 250 mm), moni-tored at 254 nm wavelength. One hundred microliters of thequenched reaction was injected to the C18 column and chroma-tography performed using 2% acetonitrile as buffer A and 80%acetonitrile as buffer B on an Agilent 1260 HPLC system. Theelution gradient profile was as follows: wash with 5 mL of bufferA, elute with a continuous linear gradient to buffer B across10 mL volume, wash with 5 mL buffer B, reduce acetonitrile con-centration to 2% using a linear concentration gradient across4 mL volume, reequilibrate with 6 mL buffer A. The flow rate was1 mL∕min throughout the procedure. Different amounts of DSAwithout the addition of the enzyme were diluted into the reactionbuffer and quenched and heated as above were injected, and astandard curve of the moles of reactant versus the area underthe elution peak was determined. As the extinction coefficientof DSA and SA can be assumed to be the same at 254-nm wa-velength, this standard curve was used to determine the numberof moles of product formed in the subsequent enzymatic reactiontime points. Measurements of the enzymatic rates were within thelinear range of substrate turnover with respect to time, which wasconfirmed by the linear correlation between the measured initialrates and enzyme concentration across four different enzymeconcentrations for each substrate concentration used. All datapoints were collected in triplicate and kinetic parameters deter-mined by fitting the data to the Michaelis–Menten equation.Steady-state kinetic parameters for DSA hydrolysis by the N220Aand K247A mutant enzymes were determined in an identicalmanner.

Hydrolysis of phenylalanine sulfamoyl adenylate (FSA) byMccF N220L/K247L was monitored in a manner identical tothe kinetics determination of DSA hydrolysis by wild-type MccF.FSA (135 μM) was incubated with 450 nM of MccF N220L/K247L enzyme at room temperature in 20 mM Hepes, pH 7.5buffer. Enzyme dilutions were made in 20 mM Hepes 100 mMKCl, pH 7.5 buffer. Reactions were initiated by the addition ofenzyme. Aliquots from the reaction mixture were taken at differ-ent time points, quenched, and substrate and products were se-parated by HPLC. Substrate and product peak elution fractionswere collected, lyophilized, dissolved in water, and analyzed byelectrospray ionization mass spectrometry to confirm the identityof the substrate and product chemical species. A calibration curvebetween number of moles of substrate and area under the sub-strate peak (without the addition of the enzyme) was generatedand used to calculate the amount of substrate consumed at dif-ferent time points during the course of the enzyme reaction.

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Fig. S1. Sequence alignment of MccF with an MccF homolog from the agrocin 84 biosynthetic gene cluster (AgnC5), hypothetical MccF homolog fromDickeya zeae, and Pseudomonas aeruginosa LD-carboxypeptidase. The catalytic triad residues are marked by star; the P1 site tryptophan residue is markedby arrowhead.

Fig. S2. Active site view of the cocrystal structure of Ser118Ala MccF with substrate McC7, illustrating the stacking interactions between Trp186 and theadenine ring of McC7. The N1 and N3 of the adenine, which have the greatest negative potential, are positioned furthest away from the six-membered ringof the indole side chain (which would bear the greatest negative potential). In addition, the positive potential bearing C8 and N9 positions of the adenine arestacked nearest to the six-membered ring. The electronegative N3 is also positioned near the basic guanidine side chain of Arg246. The two ring systems arepostulated to align so as to minimize the electrostatic repulsion between the two heterocyclic p-electron systems. Enzyme side-chain residue carbon atoms arecolored in yellow; McC7 carbon atoms are in green.

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Fig. S3. Active site view of the cocrystal structure of Trp186Phe MccF with AMP. Enzyme side-chain residue carbon atoms are colored in yellow; AMP carbonatoms are colored in green. Superimposed is a difference Fourier electron density map (contoured at 2.7σ over background in blue) calculated with coefficientsjFobsj − jFcalcj and phases from the final refined model with the coordinates of AMP deleted prior to one round of refinement. MccF N-terminal domain iscolored blue, C-terminal domain is colored pink, and the catalytic loop is colored green as in Fig. 2A.

Fig. S4. Active site view of Ser118Ala/Asn220Ala/Lys247Ala MccF. Enzyme side-chain residue carbon atoms are colored in yellow. Superimposed is a differenceFourier electron density map (contoured at 2.7σ over background in blue) calculated with coefficients jFobsj − jFcalcj and phases from the final refined modelwith the coordinates of the side chains of Ala-118, Ala-220, and Ala-247 deleted prior to one round of refinement. MccF N-terminal domain is colored blue, C-terminal domain is colored pink, and the catalytic loop is colored green as in Fig. 2A.

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Fig. S5. Electrospray ionization (ESI) mass spectrum of the substrate and product peaks for phenylalanyl sulfamoyl adenosine (FSA) cleavage by MccF Asn220-Leu/Lys247Leu double mutant enzyme. (A) ESI spectra of substrate FSA peak. Inset shows the monoisotopic masses of the major peak corresponding to FSA.(B) ESI spectra of product sulfamoyl adenosine (SA) peak. Inset shows the monoisotopic masses of the major peak corresponding to SA.

Table S1. Data collection, phasing, and refinement statistics

MccF (native) Ser118Ala MccF-ESA MccF-DSA MccF-McC7

Data collectionCell dimensionsa, b, c (Å), β(°) 54.4, 85.9, 73,1 101.2 54.5, 85.8, 73.2 101.2 54.5, 85.9, 73.1 101.2 54.4, 85.7, 72.9 101.3 54.3, 85.7, 72.9 101.3Resolution,* Å 50-1.2 (1.24-1.2) 50-1.5 (1.55-1.5) 50-1.35 (1.4-1.35) 50-1.5 (1.55-1.5) 50-1.3 (1.35-1.3)Total reflections 1,470,584 488,868 538,837 594,715 680,252Unique reflections 202,577 105,193 142,589 99,681 156,612Rsym, % 6.8 (35.2) 6.9 (12.1) 4.5 (14.5) 5.8 (24.4) 5.0 (28.2)I∕σðIÞ 38.7 (3.9) 46.3 (19.2) 39.6 (9.5) 27.9 (5.4) 29.8 (4.5)Completeness, % 98.9 (93.8) 99.7 (99.8) 98.2 (96.2) 95.6 (85.0) 97.2 (93.8)Redundancy 7.3 (5.8) 4.6 (4.6) 3.8 (3.6) 6.0 (5.3) 4.4 (4.1)RefinementResolution, Å 25-1.2 25-1.5 25-1.3 25-1.5 25-1.3No. reflections 192,327 99,890 149,510 94,666 148,363Rwork∕Rfree

† 18.3∕20.1 17.7∕19.8 17.9∕19.7 17.9∕20.2 17.4∕18.6No. of atoms

Protein 5,313 5,188 5,261 5,261 5,277Ligand — — 64 62 70Water 961 981 957 864 978

B factorsProtein 10.8 11.1 9.9 11.9 9.0Ligand — — 22.5 20.5 14.2Water 27.4 27.9 27.6 30.2 26.5

rmsdBond lengths, Å 0.005 0.006 0.005 0.006 0.005Bond angles, ° 0.97 0.984 0.987 0.986 1.000

*Highest resolution shell is shown in parenthesis.†R factor ¼ ΣðjFobsj − kjFcalcjÞ∕ΣjFobsj and R free is the R value for a test set of reflections consisting of a random 5% of the diffraction data not used inrefinement.

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