ORIGINAL ARTICLE

Role of asparaginase variable loop at the carboxyl terminalof the alpha subunit in the determination of substratepreference in plants

Michelle Gabriel • Patrick G. Telmer •

Frederic Marsolais

Received: 27 September 2011 / Accepted: 17 November 2011 / Published online: 30 November 2011

� Her Majesty the Queen in Right of Canada 2011

Abstract Structural determinants responsible for the sub-

strate preference of the potassium-independent (ASPGA1)

and -dependent (ASPGB1) asparaginases from Arabidopsis

thaliana have been investigated. Like ASPGA1, ASPGB1

was found to be catalytically active with both L-Asn and

b-Asp-His as substrates, contrary to a previous report.

However, ASPGB1 had a 45-fold higher specific activity

with Asn as substrate than ASPGA1. A divergent sequence

between the two enzymes forms a variable loop at the

C-terminal of the alpha subunit. The results of dynamic

simulations have previously implicated a movement of the

C-terminus in the allosteric transduction of K?-binding at

the surface of LjNSE1 asparaginase. In the crystal structure

of Lupinus luteus asparaginase, most residues in this segment

cannot be visualized due to a weak electron density.

Exchanging the variable loop in ASPGA1 with that from

ASPGB1 increased the affinity for Asn, with a 320-fold

reduction in Km value. Homology modeling identified a

residue specific to ASPGB1, Phe162, preceding the variable

loop, whose side chain is located in proximity to the beta-

carboxylate group of the product aspartate, and to Gly246, a

residue participating in an oxyanion hole which stabilizes

a negative charge forming on the side chain oxygen of

asparagine during catalysis. Replacement with the corre-

sponding leucine from ASPGA1 specifically lowered the

Vmax value with Asn as substrate by 8.4-fold.

Keywords Asparaginase � K?-dependent enzyme

activity � Substrate preference � Site-directed mutagenesis �Homology model � Arabidopsis

Abbreviations

ANOVA Analysis of variance

ASPG Asparaginase

ASPGA1 Asparaginase A 1 from Arabidopsis thaliana

ASPGB1 Asparaginase B 1 from Arabidopsis thaliana

EcAIII Escherichia coli isoaspartyl aminopeptidase/

asparaginase

LjNSE1 Asparaginase 1 from Lotus japonicus

LjNSE2 Asparaginase 2 from Lotus japonicus

LlA Lupinus luteus asparaginase

LB Luria-Bertani

LSD Fisher’s protected least significant difference

Ntn N-terminal nucleophile

PDB Protein data bank

TAIR The Arabidopsis information resource

Introduction

L-asparaginases (ASPGs) (EC 3.5.1.1) catalyze the hydro-

lysis of L-Asn, producing Asp and ammonia. They are

expressed in sink tissues of higher plants, where they

metabolize transported Asn (Lea et al. 2007). The plant

ASPGs are also specialized in the degradation of b-aspartyl

dipeptides (Michalska and Jaskolski 2006), arising from

the formation of isoaspartyl residues in protein, particularly

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00425-011-1557-y) contains supplementarymaterial, which is available to authorized users.

M. Gabriel � F. Marsolais

Department of Biology, University of Western Ontario,

London, ON, Canada

M. Gabriel � P. G. Telmer � F. Marsolais (&)

Southern Crop Protection and Food Research Centre,

Agriculture and Agri-Food Canada, 1391 Sandford St,

London, ON N5V 4T3, Canada

e-mail: Frederic.Marsolais@agr.gc.ca

123

Planta (2012) 235:1013–1022

DOI 10.1007/s00425-011-1557-y

during seed aging (Oge et al. 2008). Plant ASPGs belong to

the superfamily of N-terminal nucleophile (Ntn) amido-

hydrolases. Glycosylasparaginase and the Asp protease

Taspase-1 are part of the same family of Ntn amidohy-

drolases as the plant ASPGs (Oinonen et al. 1995; Guo

et al. 1998; Xuan et al. 1998; Khan et al. 2005). These

enzymes are produced as polypeptide precursors and

undergo self-cleavage, resulting in the formation of N- and

C-terminal derived a- and b-subunits, respectively. Auto-

proteolytic cleavage exposes a catalytic Thr nucleophile at

the N-terminus of the b-subunit. The mature enzyme is

a tetramer composed of two heterodimers of the a- and

b-subunit.

There are two subfamilies of ASPGs in higher plants,

corresponding to biochemical subtypes previously defined

on the basis of their dependence on K? for catalytic

activity (Sodek et al. 1980). Each subfamily is represented

by a single gene in Arabidopsis thaliana (Bruneau et al.

2006). The K?-dependent ASPGB1 (TAIR accession

number At3g16150) had 20-fold higher catalytic activity

with Asn as substrate than the K?-independent ASPGA1

(At5g08100). Activation by K? increased the specific

activity of ASPGB1 by approximately tenfold. Recently,

similar results were reported for K?-dependent and -inde-

pendent ASPGs from Lotus japonicus, LjNSE1 and -2,

respectively (Credali et al. 2011). With respect to K?,

LjNSE1 exhibits an allosteric response categorized as type

II since its activity is not absolutely dependent on mono-

valent cations (Page and Di Cera 2006). K?-activation

enhances catalytic activity and results in a slight reduc-

tion in Km for Asn (Credali et al. 2011). The K?-inde-

pendent ASPGA1 has comparable activities with Asn and

b-aspartyl dipeptides. Within the K?-independent ASPG

subfamily, enzymes from Lupinus species form a separate

clade (Bruneau et al. 2006), having a strong substrate

preference for b-aspartyl dipeptides in vitro. Lupinus luteus

LlA has an approximately 100-fold higher catalytic effi-

ciency with b-Asp-Leu as compared to Asn (Borek et al.

2004).

Structural studies of plant ASPGs and their Escherichia

coli homolog EcAIII have provided insights into the pro-

cesses of substrate binding and catalysis, autoproteolytic

cleavage and K?-activation. The crystal structure of LlA

could be easily superimposed with that of EcAIII in com-

plex with Asp (Michalska et al. 2005, 2006). In both

enzymes, a Na? ion plays a structural role and is bound by

the main chain carbonyl atoms of a subunit residues

present in a loop spanning residues Leu59 to Ile69 in L1A.

This Na? ion is distinct from the K? responsible for

ASPGB1 and LjNSE1 activation. Interestingly, mass

spectrometric analysis of ASPGB1 after extensive desalt-

ing in buffer without monovalent cations confirmed K?

binding, but suggested that the K?-dependent ASPG lacked

the Na? ion (Bruneau et al. 2006). Only half of the residues

within the Na?-binding loop of L1A are conserved in

ASPGB1. Active site residues interacting directly with Asp

belong to the b-subunit and include Arg221, which forms a

salt bridge with the a-carboxylate group, and Asp224, which

anchors the a-amino group. There is room in the substrate-

binding cavity for the second residue in the b-aspartyl

dipeptide. This end corresponds to the sugar-binding site in

glycosylasparaginase. It is lined with a-subunit residues,

Gly10, Ala12 and Gly13, forming a GAG motif. The cata-

lytic Thr193 nucleophile (position 183 in ASPGA1; Fig. 1a)

is in close proximity to the side chain carboxyl group of the

Asp product. Residues interacting with the oxygen of the

amide group of the substrate, Thr243 and Gly244, form an

oxyanion hole, stabilizing a negative charge developing

during nucleophilic attack on the amide carbon by the

Thr193 side chain and hydrolysis. The Asn66 residue, part of

the Na?-binding loop is important for the proper posi-

tioning of the Thr193 residue. Although the C-terminal half

of the a-subunit points toward the active site in the LlA

structure, the lack of electron density prevents modeling of

its last 26 residues, and thus an evaluation of its role in

substrate binding and catalysis. The sequence preceding the

autoproteolytic cleavage site, at the C-terminal end of the

a-subunit, is the most variable between the K?-dependent

and -independent ASPGs, suggesting a possible role in the

determination of substrate preference. This segment

encompasses positions 169–182 and 168–194 in Arabid-

opsis ASPGA1 and -B1, respectively (Fig. 1a).

Crystallization of a non-cleavable EcAIII mutant where

the catalytic Thr179 residue was mutated to Ala provided

insight into the mechanism of autocatalytic activation, which

involves some of the same residues involved in substrate

hydrolysis. Thr230 (residue 243 in LlA) acts as a general base,

and the side chain of Asn67 in the Na?-binding loop (residue

66 in LlA) forms an oxyanion hole with a water molecule,

stabilizing a negative charge forming on the oxygen atom of

Gly178 preceding the catalytic Thr179 nucleophile in the

cleavage intermediate (Michalska et al. 2008). In this

structure, lack of cleavage stabilized the linker region

between a- and b-subunits, and up to 11 residues forming a

loop could be visualized immediately preceding the cleavage

site. The lack of electron density for the remaining residues

highlighted the flexibility of this loop.

The structural mechanism of K?-activation was recently

uncovered through a homology modeling and site-directed

mutagenesis approach (Credali et al. 2011). LjNSE1 and -2

were modeled against the coordinates of EcAIII and LlA

(Michalska et al. 2005, 2006, 2008). Glu248, Asp285 and

Glu286 in LjNSE1 were identified as determinants of K?-

activation and putative ligands of the monovalent cation.

These residues form a negatively charged cavity at the

surface of the enzyme, at the entrance of the active site.

1014 Planta (2012) 235:1013–1022

123

According to calculations after dynamic simulations

involving charge neutralization and solvation, monovalent

cation binding to LjNSE1 appears to induce atomic fluc-

tuations of residues at the C-terminal end of the a-subunit.

These perturbations are most pronounced with Li? and

Cs?, which are the least able to activate LjNSE1. These

observations indicate that K?-activation does not involve a

direct interaction between the metal and substrate, and that

K?-binding at the protein surface triggers a conformational

change which favors substrate binding and enhances cata-

lytic activity.

Currently, there is no structural information about the

variable loop at the C-terminal end of the a-subunit from

mature plant ASPGs. In this study, K?-dependent and

-independent ASPG isoforms from Arabidopsis were

compared at the molecular level. The function of the

variable loop in the determination of substrate preference

was investigated. A residue which may contribute to the

high catalytic activity of K?-dependent ASPGs toward Asn

was identified.

Materials and methods

Structural modeling

Models of ASPGA1 and -B1 were constructed with

MODELLER, which implements comparative protein

structure modeling by satisfying spatial restraints (Eswar

et al. 2006), on the basis of coordinates from the crystal

structure of L. luteus LlA (PDB accession number 2GEZ)

(Michalska et al. 2006) and visualized with SwissPDB

viewer (Guex and Peitsch 1997). To carry out ligand

docking, Pymol was used to remove Asp from the struc-

tural model and the ligand was saved in a separate file.

Docking of Asp to ASPGB1 was performed with Auto-

Dock Vina, an automated tool designed to predict how

small molecules bind to a receptor of known three-

dimensional structure by determining the minimum inter-

action energy between the substrate and target protein

(Trott and Olson 2010). Enzyme and ligand were prepared

with AutoDock Tool, by adding all hydrogen atoms and

merging non-polar hydrogens. For Asp, all bonds were

made rotatable. A grid box centered in the active site of

ASPGB1 was defined, which was large enough to

encompass the active site and allow the ligand freedom to

move around. AutoDock Vina identified nine possible

docking conformations. These were inspected with Pymol

(Schrodinger, Cambridge, MA), and the first docking

conformation was selected on the basis of lowest energy.

Root mean square deviations and distances were calculated

with Pymol. Figures were rendered with POV-Ray

(Raytracer, Williamstown, Australia).

Generation of chimeric and deletion constructs

by recombinant PCR

Overlapping DNA sub-fragments were generated by

recombinant PCR (Higuchi 1990) using ASPGA1 and -B1

cDNAs cloned in the pCR BluntII TOPO vector (Bruneau

et al. 2006) and primers listed in Supplementary Table 1.

Forward primers contained a 50 extension complementary

to the other sequence. PCR reactions were performed using

Pfx50 DNA polymerase (Invitrogen, Burlington, ON).

Fragments longer than 100 bp were separated by agarose

gel electrophoresis and purified using the illustra GFX PCR

Fig. 1 a Amino acid sequence

alignment of K?-independent

ASPGA1, L1A and K?-

dependent ASPGB1. The arrowmarks the autoproteolytic

cleavage site giving rise to

a- and b-subunits. b Schematic

view of chimeric ASPG

enzymes

Planta (2012) 235:1013–1022 1015

123

DNA and Gel Band Purification Kit (GE Healthcare, Baie

d’Urfe, QC). Fragments shorter than 100 bp were separated

by electrophoresis on a 20% polyacrylamide gel and eluted

in 150 ll of 10 mM Tris–HCl, pH 8.0, 5 mM EDTA and

0.02% SDS, by incubation at 65�C with shaking for 16 h.

The eluate was purified with a Microspin G25 column (GE

Healthcare) and concentrated by centrifugal evaporation. A

second round of PCR was used to generate full-length

chimeric or deletion sequences. Overlapping DNA frag-

ments from the first round were added to the reaction in

equimolar amount. External primers were added to the

reaction after the second PCR cycle. Gel-purified full-

length products were cloned into the pCR4Blunt-TOPO

vector and transformed into chemically competent Esche-

richia coli TOP10F0 cells (Invitrogen). Transformed cells

were grown in Luria-Bertani (LB) medium containing

50 lg ml-1 kanamycin. Hybrid or truncated cDNAs were

sequenced with a 3130XL Genetic Analyzer (Applied

Biosystems, Streetsville, ON). Inserts derived from

ASPGA1 and -B1 coding sequences were digested at the 50

end with BamHI or KpnI, respectively, and PstI at the 30

end, and subcloned at the corresponding sites in the poly-

linker of the pQE30 vector (Qiagen, Toronto, ON) as

previously described (Bruneau et al. 2006).

Site-directed mutagenesis

Mutagenic primers were designed using the QuikChange

Primer Design Program (Agilent, Mississauga, ON) (Sup-

plementary Table 2). Mutagenized plasmids were synthe-

sized using the QuikChange II site-directed mutagenesis

kit. As much as 10 ng of plasmid containing the full-length

ASPGA1 or -B1 cDNA in pQE30 was used as template.

The presence of the mutation and integrity of the remainder

of the cDNA was verified by sequencing.

Expression of recombinant L-asparaginases

in Escherichia coli

ASPGs were expressed and purified as previously descri-

bed (Bruneau et al. 2006) with the following modifications.

After resuspension in binding buffer (50 mM sodium

phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4) cells

were lysed with a French Press. After centrifugation, the

supernatant was purified by immobilized metal affinity

chromatography on a 5 ml HisTrap Ni2?-Sepharose col-

umn using an AKTApurifier system (GE Healthcare). The

column was washed with five column volumes of wash

buffer (50 mM sodium phosphate, 500 mM NaCl, 40 mM

imidazole, pH 7.4) and eluted with a linear gradient going

from 0 to 100% of elution buffer (50 mM sodium phos-

phate, 500 mM NaCl, 500 mM imidazole, pH 7.4). Purified

protein was concentrated and desalted in 50 mM Tris–HCl,

pH 7.4, by four cycles of centrifugation and dilution using

an Amicon Ultra-15 Ultracel 30 K filter unit (Millipore,

Billerica, MA). Purified protein was stored at -80�C in

20% glycerol (v/v) after flash freezing in liquid nitrogen.

Protein quantification and SDS-PAGE were performed as

previously described (Bruneau et al. 2006).

Kinetic analysis

Spectrophotometric analyses with a coupled enzyme assay

were used to determine the kinetic parameters with Asn

(Sigma Life Sciences 11149, Oakville, ON, purity greater

than 99.5%) and b-Asp-His (Bachem Americas G-3980,

Torrance, CA, purity greater than 99%) as substrates in a

96-well format as per Bruneau et al. (2006) with modifi-

cations. The stock solutions of 100 mM Asn and 100 mM

b-Asp-His were prepared in 200 mM Tris–HCl, pH 7.0.

Asn was purified using a Dowex 1 9 8 anion exchange

column (Sigma Aldrich) to remove all traces of Asp

(Prusiner and Milner 1970). The assay buffer was 200 mM

Tris–HCl, pH 8.0, and 50 mM KCl. Absorbance readings

were taken every 5 min for a total period of 30 min. To

determine the enzyme activity, the moles of NADH

depleted were calculated using Beer’s Law. The path

length was measured to correct the calculated enzyme

activities, using the path length correction feature in KC

junior software for the Power Wave XS 96-well plate

reader (Bio-Tek Instruments, Winooski, VT). One-way

analysis of variance (ANOVA) was performed with SAS

version 9.2 (Toronto, ON).

Results

K?-dependent ASPGB1 is catalytically active

with b-Asp-His, but prefers Asn as substrate

New values are reported for the kinetic parameters of

ASPGA1 and -B1 because they were determined under

slightly different conditions from a previous study

(Table 1) (Bruneau et al. 2006). In particular, Asn was

purified by chromatography to remove any residual Asp

acting as ASPG inhibitor (Prusiner and Milner 1970).

Kinetic parameters were similar to those previously

reported, except that the Km values for Asn were higher by

three- to fourfold (Table 1). As a result, ASPGA1 had a

slight substrate preference for b-Asp-His over Asn, as

determined by a fourfold difference in catalytic efficiency

(Vmax/Km). ASPGB1 was previously reported to be specific

for Asn (Bruneau et al. 2006) and likewise LjNSE1 (Cre-

dali et al. 2011). However, assays performed at a higher

concentration of enzyme revealed that ASPGB1 was cat-

alytically active with b-Asp-His as well (Table 1). Values

1016 Planta (2012) 235:1013–1022

123

of kinetic parameters were lower than those measured with

Asn, by approximately threefold for Km, 45-fold for Vmax

and 16-fold for catalytic efficiency, indicating an overall

preference for Asn as substrate. The values of kinetic

parameters determined with b-Asp-His were relatively

similar between ASPGB1 and -A1. The main feature dif-

ferentiating the two enzymes is the higher Vmax value of

ASPGB1 with Asn as substrate.

Reciprocal exchange of the C-terminal end

of the a-subunit affects the affinity for Asn

and b-Asp-His

The sequence preceding the autoproteolytic cleavage site at

the C-terminal end of the a-subunit is the most variable

between K?-dependent and -independent ASPGs (Fig. 1a).

The variable loop itself spans positions 169–182 of

ASPGA1 and 168–194 of ASPGB1. This sequence is longer

in the K?-dependent ASPG by 13 residues. To investigate

the potential role of the variable loop in the determination of

substrate preference, reciprocal exchange of the segment

preceding the cleavage site was performed between

ASPGA1 and -B1, starting from two different positions

within the ASPG sequence: from residue 145 of ASPGA1 in

chimeras A and C, which includes a more conserved seg-

ment preceding the variable loop, and from residue 169 in

chimeras B and D, which is restricted to the variable loop

itself (Fig. 1). Chimeras A and B have the variable loop

from ASPGB1 introduced into the ASPGA1 backbone, and

reciprocally for chimeras C and D. Kinetic parameters of

the chimeric enzymes were determined with Asn and

b-Asp-His as substrates and values compared with those of

the corresponding parental, wild-type enzyme (Table 1).

In chimeras C and D, where the C-terminal end of the

a-subunit from ASPGA1 is fused to the b-subunit of

ASPGB1, autoproteolytic maturation was substantially

reduced (Fig. 2a). This was associated with a large decrease

in Vmax with either substrate, of up to a 100-fold in chimera D

(Table 1). The low catalytic activity detected is likely due to

residual cleavage in the protein extracts. The results of

kinetic analysis indicated that introduction of the shorter

variable region from ASPGA1 only had a small impact on Km

values with either substrate. In chimeras A and B, maturation

appeared relatively unaffected. Introduction of the longer

variable region from ASPGB1 into the ASPGA1 backbone

decreased the values of kinetic parameters, particularly the

Km for Asn, by up to 320-fold in chimera B. Combined with

an approximately fivefold reduction in Vmax, this resulted in a

60-fold increase in catalytic efficiency as compared to the

wild-type ASPGA1. Substrate preference was shifted in

favor of Asn over b-Asp-His, with an eightfold higher value

of catalytic efficiency. Despite the low Km value, there was

no evidence of substrate inhibition by Asn concentrations up

to 20 mM (data not shown).

Mutation of Phe162 immediately preceding the variable

loop in K?-dependent ASPGB1 specifically affects

catalytic activity with Asn

To gain insight into possible structural determinants of

substrate preference, ASPGA1 and -B1 were modeled

against the coordinates of the crystal structure of LlA

(Michalska et al. 2006) (Fig. 3a). Residues at the C-ter-

minal end of the a-subunits were excluded as they cannot

be visualized in the target and their conformation may not

be representative of the mature enzyme. Backbone atoms

Table 1 Kinetic parameters of wild-type and chimeric ASPGA1 and -B1

Asn b-Asp-His

Km (mM) Vmax (910-8

katal mg-1)

Vmax/Km (910-8

katal mg-1 mM-1)

Km (mM) Vmax (910-8

katal mg-1)

Vmax/Km (910-8

katal mg-1 mM-1)

ASPGA1 14.7 ± 1.4 1.24 ± 0.09 0.085 ± 0.003 12.6 ± 0.8 3.23 ± 0.12 0.26 ± 0.01

Chimera A 0.13 ± 0.01 0.16 ± 0.03 1.20 ± 0.24 1.01 ± 0.34 0.19 ± 0.01 0.20 ± 0.06

Chimera B 0.046 ± 0.006 0.21 ± 0.01 4.84 ± 1.34 0.52 ± 0.05 0.30 ± 0.01 0.57 ± 0.06

ANOVA p value 0.0001 0.0001 0.0002 0.0001 0.0001 0.0001

LSD 1.68 0.10 1.18 0.96 0.12 0.10

ASPGB1 6.83 ± 1.13 65.9 ± 6.0 9.72 ± 0.67 2.38 ± 0.45 1.46 ± 0.07 0.62 ± 0.09

Chimera C 20.9 ± 5.8 2.37 ± 0.34 0.12 ± 0.02 3.00 ± 0.76 0.12 ± 0.02 0.041 ± 0.010

Chimera D 4.04 ± 0.27 0.60 ± 0.03 0.15 ± 0.01 0.23 ± 0.03 0.025 ± 0.001 0.11 ± 0.01

ANOVA p value 0.002 0.0001 0.0001 0.001 0.0001 0.0001

LSD 6.85 6.87 0.77 1.02 0.085 0.11

n = 3; average ± standard deviation; analysis of variance (ANOVA)

Fisher’s protected least significant difference (LSD) at p B 0.05

Planta (2012) 235:1013–1022 1017

123

could easily be superimposed between the three structures.

The root mean square deviations between main chain

atoms were equal to 0.238 A between ASPGA1 and LlA;

0.263 A between ASPGB1 and LlA; and 0.181 A between

ASPGA1 and -B1. Asp was docked into the active site of

ASPGB1. Although the values of root mean square devi-

ations were low, they do not mean that the homology

models are accurate at this level and these remain theo-

retical. The LlA structure lacks electron density for the last

26 residues at the C-terminal end of the a-subunit, com-

mencing at position 167 in ASPGA1 (Fig. 1a). However,

structural information is available for residues immediately

preceding this segment, up to Thr166 in ASPGA1, which

are reciprocally exchanged in chimeras A and C (Fig. 1).

This segment was examined for residues variable between

ASPGA1 and -B1, which may influence the preference of

ASPGB1 for Asn as substrate. The side chain of Phe162 in

ASPGB1 was found in relative proximity to Asp bound in

the model (Fig. 3b). The nearest distance between the

aromatic ring of Phe and the proximal oxygen atom of

the carboxyl side chain of Asp was equal to 3.9 A. In

ASPGA1, the corresponding residue is Leu163 (Fig. 3c).

When Asp was positioned in a similar manner as in

ASPGB1, the nearest distance between the Leu side chain

and proximal oxygen atom of the carboxyl side chain of Asp

was equal to 4.1 A. The Phe residue is strictly conserved in

K?-dependent ASPGs, while the corresponding Leu residue

can be substituted with Val or Ile in K?-independent

ASPGs (Fig. 1a). With respect to known active site resi-

dues, Phe162 appeared close to Thr245 and Gly246 (Fig. 3d).

These residues form the oxyanion hole, which stabilizes a

negative charge forming on the oxygen atom of the amide

group of the substrate during catalysis (Michalska and

Jaskolski 2006). According to the homology model, the

nearest distance between the Phe side chain and Gly246 was

equal to 3.5, and 3.8 A for Thr245. In comparison, the

nearest distance from the Phe side chain to the hydroxyl

group of the catalytic Thr195 residue was equal to 4.2 A.

The function of several residues within or near the

variable loop was investigated by site-directed mutagene-

sis. Reciprocal mutations were performed for Phe162 and

Leu163 in ASPGB1 and -A1, respectively. A conservative

replacement of Phe162 was also performed with Trp. Arg165

in ASPGB1 and Thr166 and ASPGA1 were reciprocally

mutated as well. Contrary to Phe162 and Leu163, the side

chains of Arg165 and Thr166 point away from the substrate

in the homology models. Asn184 and Ser189, which were the

only residues strictly conserved in the variable loop of K?-

dependent ASPGs, whose side chains could participate in

hydrogen bonding, were also mutated (Fig. 1a). Overall,

the mutations had relatively modest effects on kinetic

parameters (Table 2). The largest effect was observed in

the F162L mutant, with an 8.4-fold decrease in Vmax value

with Asn, whereas the Vmax with b-Asp-His was similar to

that of the wild-type enzyme (Table 2). The decrease in

Vmax value with Asn was intermediate in F162W, of

approximately fourfold. Introduction of the more bulky

residues in ASPGA1 mutants, L163F and T166R, only

affected Km and Vmax values with b-Asp-His and not those

with Asn. With b-Asp-His, the Vmax value of the L163F

mutant was reduced by approximately fivefold and the Km

value by twofold. Replacement of Asn184 or Ser189 with

Ala resulted in a small decrease in Vmax value with Asn as

substrate, of approximately threefold (Table 2). When the

functional group of the side chain was still present in

conservative mutants N184Q and S189T, the Vmax value

Fig. 2 SDS-PAGE of wild-type and chimeric enzymes (a) site-

directed (b) and deletion mutants (c). The position of molecular

weight markers is indicated on the left. p polypeptide precursor; a and

b indicate subunits

1018 Planta (2012) 235:1013–1022

123

with Asn as substrate was similar or slightly higher than

that of the wild-type ASPGB1.

The variable region of ASPGB1 tolerates small

deletions which impact self-cleavage and catalytic

activity

It is possible that the large decrease in Km value for Asn in

chimera A, as compared with the parental ASPGA1, may

be due to the presence of a longer variable loop at the

C-terminal end of the a-subunit, derived from ASPGB1. To

test this hypothesis, small truncations of the variable region

were introduced in chimera A and in ASPGB1 (Table 3).

Kinetic parameters were determined with Asn and b-Asp-

His and values were compared with those of the corre-

sponding parental enzyme. Several of the deletions were

detrimental to autoproteolytic cleavage of the polypeptide

precursor (Fig. 2c). Individual deletions had opposite

effects on self-cleavage of ASPGB1 and chimera A, con-

sistent with the chimeric enzyme having the opposite

backbone derived from ASPGA1. Although two of the

deletions raised the Km value of chimera A mutants with

Asn, they were far from restoring this value to the level

observed with the parental ASPGA1 (Table 3).

Discussion

A reexamination of the substrate specificity of the K?-

dependent ASPGB1 revealed that the enzyme accepted an

isoaspartyl dipeptide as substrate, contrary to what was

previously reported (Bruneau et al. 2006). The catalytic

activity of ASPGB1 with b-aspartyl dipeptides was actu-

ally expected if one considered the high degree of sequence

identity with ASPGA1 (55%) and the similarity of their

structural models (Fig. 3a) (Michalska and Jaskolski 2006).

ASPGB1 displays a substrate preference for Asn over

b-Asp-His, mainly due to a higher catalytic activity. This

high catalytic activity with Asn is the main feature

differentiating the K?-dependent ASPGB1 from the

K?-independent ASPGA1.

The sequence at the C-terminal end of the a subunit is

highly variable between K?-dependent and -independent

ASPGs. Dynamic simulations involving charge neutraliza-

tion with different monovalent cations and solvation of K?-

dependent LjNSE1 have previously indicated a high degree

of atomic fluctuations of residues present in this region,

located approximately between positions 160–195 of

ASPGB1 (Fig. 1a) (Credali et al. 2011). Although pertur-

bations were still present, they were minimized in the

presence of K?, which provided the highest degree of

enzyme activation. These results suggested that allosteric

activation mediated by K? was transduced by a change in

the conformation of the C-terminal end of the a subunit. In

support of this hypothesis, K? appears coordinated by res-

idues located at the entrance of the active site, and therefore

activation does not appear to involve a direct interaction

between K? and the substrate (Credali et al. 2011).

The lack of information on the conformation of the

variable loop at the C-terminal end of the a subunit from

Fig. 3 Molecular modeling of

ASPGA1 and -B1. Homology

models were constructed with

MODELLER on the basis of

coordinates from the crystal

structure of L1A (Michalska

et al. 2006). The Asp molecule

docked into the ASPGB1

structure using AutoDock Vina.

a Superposition of LlA (green),

ASPGA1 (blue) and ASPGB1

(red). The Asp molecule is

shown in black. b Interaction

between Phe162 and the side

chain carboxyl group of Asp in

ASPGB1. c The corresponding

residue in ASPGA1 is Leu163.

d Phe162 in ASPGB1 is located

in proximity to Gly246, which

participates to the oxyanion hole

stabilizing a negative charge on

the oxygen atom of the amide

group during catalysis

Planta (2012) 235:1013–1022 1019

123

the crystal structures of mature LlA and EcAIII hampers an

interpretation of its role in substrate binding or catalysis.

The lack of electron density highlights the flexibility of the

loop region. Residues which can be modeled point toward

the active site (Michalska et al. 2006), and it is possible

that after internal cleavage, the C-terminal end of the asubunit remains in proximity to the substrate-binding

cavity (Credali et al. 2011). Exchange of segments which

Table 2 Effect of mutations at the C-terminal end of the a subunit on kinetic parameters of wild-type ASPGA1 and -B1

Asn b-Asp-His

Km (mM) Vmax (910-8

katal mg-1)

Vmax/Km (910-8

katal mg-1 mM-1)

Km (mM) Vmax (910-8

katal mg-1)

Vmax/Km (910-8

katal mg-1 mM-1)

ASPGA1 14.7 ± 1.4 1.24 ± 0.09 0.085 ± 0.003 12.6 ± 0.8 3.23 ± 0.12 0.26 ± 0.01

ASPGA1-L163F 12.7 ± 1.6 1.12 ± 0.17 0.088 ± 0.007 6.79 ± 1.39 0.57 ± 0.02 0.085 ± 0.016

ASPGA1-T166R 14.3 ± 1.1 0.94 ± 0.05 0.066 ± 0.002 3.13 ± 0.31 1.12 ± 0.04 0.36 ± 0.03

ANOVA p value n.s. n.s. 0.003 0.0001 0.0001 0.0001

LSD 2.8 0.22 0.009 1.86 0.13 0.037

ASPGB1 6.83 ± 1.13 65.9 ± 6.0 9.72 ± 0.67 2.38 ± 0.45 1.46 ± 0.07 0.62 ± 0.09

ASPGB1-F162 W 5.12 ± 0.37 18.0 ± 0.95 3.52 ± 0.074 3.90 ± 0.33 0.82 ± 0.07 0.21 ± 0.04

ASPGB1-F162L 3.25 ± 0.36 7.84 ± 0.31 2.41 ± 0.23 5.56 ± 0.45 1.38 ± 0.04 0.25 ± 0.01

ASPGB1-R165T 10.7 ± 3.1 20.7 ± 2.8 1.93 ± 0.32 5.62 ± 0.29 1.82 ± 0.12 0.32 ± 0.01

ASPGB1-N184Q 10.5 ± 3.0 105 ± 19 10.0 ± 1.4 3.28 ± 0.21 1.98 ± 0.22 0.60 ± 0.11

ASPGB1-N184D 4.11 ± 0.21 25.3 ± 0.7 5.14 ± 0.46 4.15 ± 0.42 0.78 ± 0.04 0.19 ± 0.01

ASPGB1-N184A 4.53 ± 0.42 28.2 ± 5.0 6.22 ± 0.53 2.90 ± 0.06 1.44 ± 0.02 0.50 ± 0.02

ASPGB1-S189T 3.25 ± 0.20 58.2 ± 1.6 17.9 ± 0.64 6.65 ± 0.29 2.04 ± 0.08 0.31 ± 0.01

ASPGB1-S189C 4.72 ± 0.41 23.0 ± 1.6 5.22 ± 0.44 3.56 ± 0.04 3.69 ± 0.01 1.04 ± 0.01

ASPGB1-S189A 4.23 ± 0.28 22.6 ± 1.3 5.34 ± 0.06 4.91 ± 0.37 3.20 ± 0.18 0.65 ± 0.02

ANOVA p value 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001

LSD 2.48 11.5 1.02 0.55 0.18 0.08

n = 3; average ± standard deviation

n.s. not significant

Table 3 Effect of deletions at the C-terminal end of the a subunit on kinetic parameters of chimera A and wild-type ASPGB1

Asn b-Asp-His

Km (mM) Vmax (910-8

katal mg-1)

Vmax/Km (910-8

katal mg-1 mM-1)

Km (mM) Vmax (910-8

katal mg-1)

Vmax/Km (910-8

katal mg-1 mM-1)

Chimera A 0.13 ± 0.01 0.16 ± 0.03 1.20 ± 0.24 1.01 ± 0.34 0.19 ± 0.01 0.20 ± 0.01

Chimera A D169–173 0.26 ± 0.01 0.082 ± 0.001 0.32 ± 0.01 0.67 ± 0.06 0.13 ± 0.01 0.19 ± 0.01

Chimera A D169–178 0.70 ± 0.07 0.71 ± 0.01 1.02 ± 0.08 n.d. n.d. n.d.

Chimera A D181–184 0.19 ± 0.02 0.55 ± 0.02 2.90 ± 0.22 0.083 ± 0.007 0.59 ± 0.01 7.16 ± 0.52

Chimera A D181–189 0.94 ± 0.08 0.18 ± 0.01 0.20 ± 0.02 0.26 ± 0.01 0.13 ± 0.01 0.51 ± 0.03

ANOVA p value 0.0001 0.0001 0.0001 0.0008 0.0001 0.0001

LSD 0.08 0.03 0.27 0.33 0.01 0.49

ASPGB1 6.83 ± 1.13 65.9 ± 6.0 9.72 ± 0.67 2.38 ± 0.45 1.46 ± 0.07 0.62 ± 0.09

ASPGB1 D169–173 2.63 ± 0.68 19.1 ± 1.6 7.27 ± 1.36 3.77 ± 0.72 1.83 ± 0.31 0.49 ± 0.16

ASPGB1 D169–178 3.95 ± 0.43 7.26 ± 0.14 1.84 ± 0.17 3.80 ± 0.64 0.19 ± 0.02 0.049 ± 0.003

ASPGB1 D181–184 3.12 ± 0.43 6.67 ± 0.35 2.14 ± 0.20 1.03 ± 0.03 0.37 ± 0.01 0.36 ± 0.01

ASPGB1 D181–189 2.60 ± 0.26 3.45 ± 0.13 1.32 ± 0.10 2.29 ± 0.63 0.088 ± 0.014 0.039 ± 0.005

ANOVA p value 0.0001 0.0001 0.0001 0.0005 0.0001 0.0001

LSD 1.20 5.02 1.25 1.00 0.26 0.15

n = 3

n.d. not determined, did not display saturable kinetics with b-Asp-His

1020 Planta (2012) 235:1013–1022

123

include the variable loop affects autoproteolytic cleavage

in chimeras C and D having an ASPGB1 backbone. In this

case, effects on the reaction rate could not be interpreted as

they likely reflected reduced levels of the cleaved, mature

enzyme. Chimeras A and B, having an ASPGA1 backbone,

retained a relatively normal cleavage into subunits. Intro-

duction of a central segment with 13 additional residues

from ASPGB1 led to an increased affinity of the enzymes

for their substrate, particularly Asn. These results suggest

that the variable loop at the C-terminal end of the a subunit

may be close to the active site in the mature enzyme.

Exchange of a longer segment in chimera A had less

impact on kinetic parameters than in chimera B, suggesting

that the orientation of the variable loop was important and

that the inclusion of a longer segment preserved a more

favorable conformation. The fact that internal deletions in

the variable loop could be tolerated and some level of

catalytic activity preserved is consistent with the variable

length of this interval even within members of the same

ASPG subfamily. Starting from residue 160 in ASPGA1,

the length of the variable loop ranges from 19 to 44 resi-

dues in ASPG sequences included in the phylogenetic tree

in Bruneau et al. (2006) (Fig. 1a). These results highlight

that besides catalytic activity, autoproteolytic maturation

imposes a severe constraint over sequence variability in the

linker between a and b subunits.

Site-directed mutations at the C-terminal end of the asubunit had relatively small effects on kinetic parameters.

The most pronounced effect was an 8.4-fold decrease in

Vmax with Asn of the F162L mutant. Homology modeling

and ligand docking identified that Phe162 was located in

proximity to Asp and several active site residues. Although

the distance predicted is relatively high at 3.5 A, it is

possible that the Phe side chain acts to position the Gly246

residue part of the catalytic oxyanion hole. Alternatively, it

is possible that the C-terminus of the a-subunit affects the

overall structure of the enzyme, and that the F162L

mutation indirectly introduces a small modification in

the active site, leading to the decreased catalytic activity

with Asn. The former hypothesis implies that Asn and

b-aspartyl dipeptides are positioned slightly differently at

the active site of ASPGB1. As compared to Asn, the ori-

entation of b-aspartyl dipeptides may be influenced by

interactions with residues belonging to the GAG motif

equivalent to the sugar-binding site in glycosylasparagin-

ase (Michalska et al. 2006). In ASPGA1, the L163F

mutation only affected the kinetic parameters with b-Asp-

His and not with Asn, suggesting that the Phe side chain

interferes with binding of the b-histidyl moiety. In con-

clusion, the C-terminal end of the a subunit may contain

structural determinants which influence, directly or indi-

rectly, the substrate preference of plant K?-dependent

ASPGs. This hypothesis is consistent with the results of

dynamic simulations linking allosteric activation by K?

with atomic fluctuations of residues within this segment

(Credali et al. 2011). The availability of a crystal structure

for a K?-dependent ASPG would provide additional

insight into the structural determinants of substrate pref-

erence in plant ASPGs.

Acknowledgments We are indebted to the staff at the Southern

Crop Protection and Food Research Centre, Ida van Grinsven for

DNA sequencing and Alex Molnar for preparation of figures, and

thank Donald B. Hayden from the Department of Biology, University

of Western Ontario, for acting as MG’s co-supervisor during her

honors’ thesis project. This work was supported by the Discovery

Program of the Natural Sciences and Engineering Research Council.

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