doi:10.1016/j.jmb.2010.03.042 J. Mol. Biol. (2010) 398, 703–714

Available online at www.sciencedirect.com

Crystal Structure of the Leucine Aminopeptidase fromPseudomonas putida Reveals the Molecular Basis forits Enantioselectivity and Broad Substrate Specificity

Avinash Kale1, Tjaard Pijning1, Theo Sonke2, Bauke W. Dijkstra1

and Andy-Mark W. H. Thunnissen1⁎

1Laboratory of BiophysicalChemistry, GroningenBiomolecular Sciences andBiotechnology Institute,University of Groningen,Nijenborgh 4, 9747 AGGroningen, The Netherlands2DSM PharmaceuticalProducts - Innovative Synthesis& Catalysis, P.O. Box 18, 6160MD Geleen, The Netherlands

Received 30 November 2009;received in revised form17 March 2010;accepted 20 March 2010Available online30 March 2010

*Corresponding author. E-mail adda.m.w.h.thunnissen@rug.nl.Present address: A. Kale, Departm

Biology & Biotechnology, UniversityCourt, Western Bank, Sheffield S10Abbreviations used: ppLAP, Pseud

aminopeptidase; LAP, leucine aminEscherichia coli leucine aminopeptidalens leucine aminopeptidase; rmsd,deviation; LPA, L-leucinephosphoni

0022-2836/$ - see front matter © 2010 E

The zinc-dependent leucine aminopeptidase from Pseudomonas putida(ppLAP) is an important enzyme for the industrial production ofenantiomerically pure amino acids. To provide a better understanding ofits structure–function relationships, the enzyme was studied by X-raycrystallography. Crystal structures of native ppLAP at pH 9.5 and pH 5.2,and in complex with the inhibitor bestatin, show that the overall folding andhexameric organization of ppLAP are very similar to those of the closelyrelated di-zinc leucine aminopeptidases (LAPs) from bovine lens andEscherichia coli. At pH 9.5, the active site contains two metal ions, oneidentified as Mn2+ or Zn2+ (site 1), and the other as Zn2+ (site 2). By using ametal-dependent activity assay it was shown that site 1 in heterologouslyexpressed ppLAP is occupied mainly by Mn2+. Moreover, it was shown thatMn2+ has a significant activation effect when bound to site 1 of ppLAP. AtpH 5.2, the active site of ppLAP is highly disordered and the two metal ionsare absent, most probably due to full protonation of one of the metal-interacting residues, Lys267, explaining why ppLAP is inactive at low pH. Astructural comparison of the ppLAP-bestatin complex with inhibitor-boundcomplexes of bovine lens LAP, along with substrate modelling, gave clearand new insights into its substrate specificity and high level of enantios-electivity.

© 2010 Elsevier Ltd. All rights reserved.

Keywords: leucine aminopeptidase; X-ray crystallography; di-zinc pro-teases; substrate specificity; enantioselectivity

Edited by M. Guss

Introduction

Aminopeptidases are metalloproteinases thatcleave N-terminal residues from proteins andsmall oligopeptides. These enzymes are widelydistributed in nature and play crucial roles inseveral important physiological processes, includingprotein degradation and turnover, protein matura-

ress:

ent of Molecularof Sheffield, Firth

2TN, UK.omonas putida leucineopeptidase; ecLAP,se, blLAP, bovineroot-mean-squarec acid.

lsevier Ltd. All rights reserve

tion, the metabolism of biologically active peptidesand antigen presentation.1,2 Aminopeptidases haveattracted additional interest due to their applicabil-ity for the production of peptides and amino acidsused in the food, agrochemical and pharmaceuticalindustries.3-6 An example of such an industrialenzyme is the leucine aminopeptidase from Pseudo-monas putida ATCC 12633 (ppLAP), which has alongstanding use as a whole-cell biocatalyst for theenantioselective hydrolysis and enzymatic resolu-tion of a broad range of DL-amino acid amideracemates.7,8 PpLAP is a member of the M17 familyof di-zinc-dependent leucine aminopeptidases(LAPs; EC 3.4.11.1 and EC 3.4.11.10),9,10 whichalso includes the well studied LAPs from bovinelens (blLAP) and Escherichia coli (ecLAP, also knownas PepA). X-ray crystallographic analysis of blLAPand ecLAP, which share a level of sequence identitywith ppLAP of 31% and 53%, respectively, hasprovided important insights into the structure and

d.

Table 1. Data collection and refinement statistics ofppLAP

Bestatin-boundHigh pH(pH 9.5)

Low pH(pH 5.2)

A. Data collectionBeam line

(ESRF)ID14-1 ID23-2 ID14-3

Wavelength (Å) 0.9340 0.8726 0.9300Space group P1 P1 P63Unit cell parameters

a (Å) 95.9 95.8 116.9b (Å) 95.9 95.9 116.9c (Å) 96.0 96.3 137.9α (°) 100.8 68.4 90β (°) 107.8 76.3 90γ (°) 93.2 94.9 120

Highestresolution (Å)

1.50 2.20 2.75

No measuredreflections

941,319 205,478 111,116

No uniquereflections

484,976 166,778 27,831

Completeness(%)

93.0 (92.5) 99.1 (96.1) 100 (99.5)

Rmerge 0.054 (0.194) 0.044 (0.372) 0.033 (0.353)Mean I/σI 15.0 (3.6) 20.2 (2.4) 39.7 (3.8)

B. RefinementResolution

range (Å)94 – 1.50 91 – 2.20 58 – 2.75

Rwork 0.149 0.192 0.212Rfree 0.173 0.251 0.267Overall

B-factor (Å2)12.9 13.7 32.1

Composition ofasymmetricunit

Six polypeptidechains (residues1–497), 6 Zn2+,6 Mn2+, 6 K+,6 bicarbonateions 6 bestatininhibitors,2974 watermolecules

Six polypeptidechains (residues1–497), 6 Zn2+,6 Mn2+, 6 K+,6 bicarbonateions 702 water

molecules

Twopolypeptide

chains(residues1–146,150–269,291–497)

rmsd from idealBond lengths

(Å)0.015 0.015 0.016

Bond angles (°) 1.5 1.5 1.7Ramachandran plot

Mostfavoured (%)

98.2 97.2 98.0

Additionallyallowed (%)

1.8 2.8 2.0

MolprobityScore 1.51 2.0 1.95

Data in parentheses are for the highest resolution shell.Rwork=Σhkl||Fobs|–|Fcalc||/Σhkl|Fobs|, where the crystallo-graphic R-factor was calculated with 95% of the data used in therefinement. Rfree is the crystallographic R-factor based on 5% of

704 Structure of P. putida Leucine Aminopeptidase

catalytic mechanism of M17 LAPs.11-16 In particular,on the basis of the crystal structures of blLAP boundwith inhibitors and transition-state analogues likebestatin and L-leucinephosphonic acid, the LAPresidues with proposed roles in catalysis, in coordi-nating the zinc ions, and/or binding substrate wereidentified.Knowledge of the biochemical, catalytic, and

structural properties of ppLAP is important toimprove its effectiveness as an industrial enzyme.Like its homologues, ppLAP requires the presenceof divalent metal ions for its activity, in particularZn2+ and/or Mn2+. It displays clear amide-hydro-lysing activity between pH 7 and pH 11, but isinactive at pH 6 or lower.6,7 Dipeptides arehydrolysed as well as single amino acid amides,with a clear preference for substrates with ahydrophobic side chain at their N-terminus. Sub-strates with an N-terminal leucine residue are mostreadily hydrolysed, but significant activity is foundwith substrates with an N-terminal methionine,phenylalanine, or isoleucine residue. In addition, avariety of non-proteinogenic amino acid amideswith different hydrophobic side chains, such asphenylglycine amide and various allylglycineamides, form good ppLAP substrates.6 In contrast,peptides and amides with small or negativelycharged N-terminal amino acid residues, such asglycine, alanine, serine, valine, aspartic acid andglutamic acid, are poor substrates. Because itsactivity requires that the chiral Cα atom has oneproton substituent, α,α-disubstituted amino acidamides like DL-α-methyl-valine amide, are nothydrolysed. Finally, the enzyme is highly enantio-selective towards substrates that have an S-configuration at their N-terminal chiral Cα atom(i.e., L-amino acid amides).6,7

To provide an accurate structural model forexplaining the biochemical and catalytic propertiesof ppLAP, we have analysed this enzyme by X-raycrystallography. Here, we report a high-pH and alow-pH crystal structure of unliganded ppLAPdetermined at 2.2 Å resolution and at 2.75 Åresolution, respectively. In addition, we describethe high-resolution crystal structure of a ppLAP–bestatin complex determined at 1.5 Å resolution.Analysis of these structures, along with substratemodelling studies, allowed us to provide newinsights into the structural and functional featuresof ppLAP.

the data selected at random and withheld from the refinement forcross-validation.

Results

Overall structure

Crystal structures of ppLAP were elucidated to aresolution of 2.75 Å (pH 5.2, unliganded), 2.2 Å(pH 9.5, unliganded), and 1.5 Å (pH 7.5, withbound bestatin inhibitor) (see Table 1 for thecrystallographic statistics). The overall features ofthe ppLAP structure are identical in all three

crystal forms and structural differences are re-stricted mainly to the active site region. Thecrystals reveal the presence of a ppLAP hexamerthat is highly similar to the ecLAP and blLAPhexamers (Fig. 1a). In solution, ppLAP exists alsoas hexamers, which was evident from gel-filtrationand dynamic light-scattering analysis (data notshown). The subunits that form the hexamercontain two domains with mixed α/β structurethat are linked by a long α-helix (Fig. 1b). The

Fig. 1. Overall structure of ppLAP. (a) Ribbon representation of the ppLAP hexamer (high-pH structure) with eachchain in a different colour and viewed along the 3-fold rotation axis. The helices that connect the two domains in eachsubunit are indicated with darker colours. The overall shape of the hexamer is triangular with an edge length ofapproximately 120 Å and a maximum thickness of approximately 85 Å. (b) Ribbon diagram of a single ppLAP subunit(high-pH structure) indicating the two domains (N-domain, N-terminal domain; C-domain, C-terminal domain) and thethree metal ions (M1, site-1 metal; M2, site-2 metal; M3, site-3 metal). The helix that connects the two domains is indicatedwith dark grey. The region of the active site that is highly disordered in the low-pH structure is shown in red.

705Structure of P. putida Leucine Aminopeptidase

smaller of the two domains, the N-terminaldomain (residues 1–164), is composed of a six-stranded mixed parallel/anti-parallel β-sheetflanked by several α-helices on both sides. TheC-terminal domain (residues 193–497) contains acentral eight-stranded mixed parallel/anti-parallelβ-sheet, surrounded by α-helices on both sides anda small three-stranded β-sheet involved in oligo-merization. The long α-helix that connects the N-and C-terminal domains comprises residues 165–192. The C-terminal domain contains the active siteand shows the highest degree of similarity withthe other LAP structures, both in sequence and inthree-dimensional structure. Domain superposi-tions of the high-pH structure of ppLAP withecLAP and blLAP reveal a root-mean-squaredeviation (rmsd) in Cα positions of 0.6 Å (304residues, 63% sequence identity) and 1.3 Å (302residues, 42% sequence identity), respectively. TheN-terminal domain of ppLAP is highly conserved,but less than the C-terminal domain, with rmsdvalues of 1.5 Å (ecLAP, 161 residues, 35% sequenceidentity) and 2.5 Å (blLAP, 132 residues, 18%sequence identity), respectively. Like the otherLAPs, the six protomers in the ppLAP hexamerform a dimer of trimers with 32 symmetry. The C-terminal domains are at the core of the hexamer,where they pack around the central 3-fold axis andstabilise the trimer-to-trimer packing. The N-terminal domains form the corners of the triangu-lar-shaped hexamer and further stabilise thetrimer-to-trimer packing by making dimeric inter-actions with each other.

Structure of unliganded ppLAP at pH 9.5

In the high-pH crystal structure three metal ionsare bound to each protomer in the hexamer (Fig. 1b).Two metal-binding sites are located in the active site(Fig 2a; Supplementary Data Fig. S1A), similar toblLAP and ecLAP. Previously, it was shown forblLAP that one of these metal-binding sites (site 1)allows exchangeable binding of various divalentmetal cations (e.g., Zn2+, Mn2+, Mg2+ and Co2+),whereas the other metal-binding site (site 2) isspecific for either Zn2+ or Co2+ and its bound ioncannot be readily exchanged.12,17 X-ray fluorescenceanalysis of ppLAP at beamline ID29 of the ESRF,Grenoble, revealed the presence of zinc and man-ganese in the high-pH ppLAP crystals (Fig. 2b). Onthe basis of that analysis and the high degree ofstructural similarity of ppLAP with blLAP, weexpect the non-exchangeable metal site 2 in ppLAPto be fully occupied by a Zn2+ and the exchangeablemetal site 1 by Mn2+ or a mixture of Mn2+ and Zn2+.Figure 2a shows the geometry of metal-binding

sites 1 and 2 of ppLAP. All residues that coordinatethe two metal ions are identical with those foundin the active sites of ecLAP and blLAP, and thecoordinating bond distances and metal-to-metaldistances are very similar to those reported for thehomologous LAP structures. The site-1 Mn2+/Zn2+

and site-2 Zn2+ are both pentacoordinated in adistorted pyramidal coordination geometry. Themetal-coordinating atoms are mostly carboxylateoxygens from the side chains of three aspartateand one glutamate residue (Asp272, Asp290,

Fig. 2. Coordination geometryand identity of the two active sitemetal ions in the high-pH ppLAPstructure. (a) Stereo diagram of theactive site in a subunit of ppLAP,showing the two metals (M1 andM2) and the coordinating residues(in ball-and-sticks). Also shown arethe metal-bridging water molecule(red sphere), and the bicarbonatemolecule (BCT). Broken lines indi-cate the metal-coordinating bondsand the hydrogen bond betweenBCT and the metal-bridging watermolecule. (b) X-ray fluorescence(emission) spectra of a ppLAP crys-tal grown at pH 9.5 and at pH 5.2,measured at the ID29 beamline ofthe ESRF. The Kα and Kβ emissionlines of Mn and Zn are indicated(5.9 and 6.5 keV for Mn, 8.6 and9.6 keV for Zn).

706 Structure of P. putida Leucine Aminopeptidase

Asp349 and Glu351). In addition, the site-1 Mn2+/Zn2+ is coordinated by the main chain carbonyloxygen of Asp349, and the site-2 Zn2+ makes abond with the ε-amino group of a lysine residue(Lys267). A water molecule or hydroxide ion isobserved at a bridging position, binding to bothmetal ions simultaneously, as was observed in theunliganded structures of blLAP and ecLAP.13,15

The high-pH structure of ppLAP also shows thepresence of a bicarbonate ion bound to the activesite, at a position identical with that observed inblLAP and ecLAP. The bicarbonate ion is bound toArg353 and makes a hydrogen bond to the metal-bridging water molecule/hydroxide ion.The third metal ion bound in ppLAP is located at

the C-terminal end of the inter-domain linker helix(Fig. 1b). This metal-binding site 3 has so far beenidentified only in blLAP.13,14 The coordinationgeometry and relatively long metal–ligand bonddistances are most suited for a monovalent sodiumor potassium ion. Because the crystallization proce-

dure of ppLAP involved the presence of potassium,it appears likely that a K+ is bound to metal-bindingsite 3 of ppLAP, which was confirmed by a B-factoranalysis (not shown). As discussed for blLAP,14 therole of the metal ion in site 3 is unclear. Most likely ithas a structural role stabilizing the interface betweenthe linker helix and the C-terminal domain.

Active site metal composition andmetal-dependent activity

To better define the metal composition of site 1in the ppLAP structure and analyse the effect oncatalysis when either Zn2+ or Mn2+ occupies thissite, the activity of purified ppLAP used forcrystallization was compared to the activities ofEDTA-treated ppLAP for which the metal in site 1was fully replaced by either Zn2+ or Mn2+ (Fig.3a). The results indicate that ppLAP is significantlyless active when site 1 is occupied with Zn2+ thanwhen it is occupied with Mn2+. This was

Fig. 3. Metal-dependent activity of ppLAP. (a) Activity profile of ppLAP for differently treated samples: untreated,after purification; EDTA, after treatment with EDTA to remove the M1 metal; Mn2+, after replacing the M1 metal withMn2+, resulting in (Mn2+-Zn2+)-bound enzyme; Zn2+, after replacing the M1 metal with Zn2+, resulting in (Zn2+-Zn2+)-bound enzyme. Activity is expressed in arbitrary units relative to that of untreated enzyme. (b) Activity profile of ppLAPafter adding Zn2+ (ZZ) or Mn2+ (ZM) to previously prepared (Zn2+-Zn2+)-bound enzyme. Activity is expressed inarbitrary units relative to that of (Zn2+-Zn2+)-bound enzyme (Z). (c) Activity profile of ppLAP after adding Mn2+ (MM) orZn2+ (MZ) to previously prepared (Mn2+-Zn2+)-bound enzyme. Activity is expressed in arbitrary units relative to that of(Mn2+-Zn2+)-bound enzyme (M). Error bars are estimated from multiple measurements.

707Structure of P. putida Leucine Aminopeptidase

confirmed by competitive activation/inhibitionexperiments in which the activity of (Zn2+-Zn2+)-bound and (Mn2+-Zn2+)-bound ppLAP (referring tothe metals occupying sites 1-2) was analysed afterthe addition of a 17-fold excess of either Zn2+ orMn2+ (Fig. 3b and c). Addition of Zn2+ to (Zn2+-Zn2+)-bound ppLAP or Mn2+ to (Mn2+-Zn2+)-bound ppLAP did not significantly affect theenzyme activity (the small decrease in activitycan be attributed to measurement errors and/orinstability of the enzyme). However, addition ofMn2+ to (Zn2+-Zn2+)-bound ppLAP resulted in a∼2.5-fold increase of activity, and addition of Zn2+

to (Mn2+-Zn2+)-bound ppLAP caused a ∼70%decrease in activity. Purified protein isolatedfrom the E. coli cytoplasm, which was used forthe crystallizations, has an activity that is compa-rable to that of treated ppLAP with Mn2+ in site 1,indicating that in the high-pH ppLAP structure site1 is predominantly occupied by Mn2+.

Structure of unliganded ppLAP at pH 5.2

In contrast to the high-pH ppLAP structure, theactive site region is highly disordered and theactive site metals are absent from the low-pHstructure of ppLAP (Figs. 1b and 2b). The loss ofthe two metals from the active site at pH 5.2 ismost likely the result of Lys267 being in a fullyprotonated state, and therefore unsuitable to serve

as coordinating ligand for the site-2 metal ion. Thiswould explain also why ppLAP is inactive at pH 6or below.6 Partial protonation of some of themetal-coordinating carboxylate groups might fur-ther destabilize metal binding. A large segment ofthe active site in the low-pH ppLAP structure,residues 270 – 290, is not visible in the 2Fo – Fc andFo – Fc electron density maps (Fig. 1b). Thissegment contains two of the metal-coordinatingligands and its disorder in the low-pH ppLAPstructure signifies the importance of the metals formaintaining the integrity of the active site.

Structure of bestatin-bound ppLAP

Highly ordered and well diffracting crystals ofbestatin-bound ppLAP were obtained from proteinsubsequently treated with EDTA and Mn2+ toensure site 1 was fully occupied by Mn2+. TheppLAP-bestatin crystal structure showed excellentdensity for the bestatin inhibitor in the active site(Supplementary Data Fig. S1B). Structural represen-tations of the binding mode of bestatin are providedin Supplementary Data Fig. S2. No significantdifference was observed in the positions of residuesand metal ions in the active site when comparing thebestatin-bound ppLAP structure with the unli-ganded, high-pH structure. The binding interactionsof bestatin in the active site of ppLAP are verysimilar to those reported for the blLAP complexes

708 Structure of P. putida Leucine Aminopeptidase

with bestatin,11,18 and the bestatin derivative micro-ginin FR1.19 A schematic overview of the polarinteractions of bestatin with ppLAP is given in Fig. 4.The most conspicuous interaction is the replacementof the bridging water/hydroxide ion between thetwo active site metal ions by the hydroxyl group ofbestatin. Two additional metal-coordinating bondsare formed by the inhibitor, between the terminalamino group and site-2 Zn2+ and between thepeptide carbonyl group and site-1 Mn2+, such thatboth metals are 6-fold coordinated in an octahedralgeometry. The D-phenylalanine side chain binds inthe hydrophobic S1 pocket (following the nomen-clature of protease sub-sites in Ref. 20) and isstabilized by van der Waals interactions withMet287, Thr376, Ile382, Ala466 and Trp470. TheL-leucyl side chain binds in the S1′ subsite makingvan der Waals contacts with Ala350, Asn347 andLeu377, while the terminal carboxylate group ismore solvent-exposed, forming one hydrogen bondwith the main chain amide of Gly379.

Comparison of bestatin-bound ppLAP withL-leucinephosphonic acid-bound blLAP

Bestatin is not a true transition state analogue ofppLAP, and therefore one may expect differencesbetween its binding mode and that of ppLAPsubstrates. In particular, in bestatin the chiral C3carbon atom to which the terminal amino groupand phenylalanine side chain are attached (the P1

residue of the inhibitor) has an R-configuration,but the equivalent Cα atom of the ppLAP peptidesubstrates has an S-configuration (SupplementaryData Fig. S3). In addition, bestatin contains amethyl hydroxyl group, inserted between thechiral C3 carbon atom and the peptide bond,which is not present in the normal ppLAPsubstrates. To investigate the interaction ofppLAP with natural substrates, the ppLAP-bestatinstructure was superimposed on the structure ofblLAP complexed with L-leucinephosphonic acid(LPA) (Fig. 5). This latter complex is considered toclosely resemble the presumed tetrahedral gem–diolate transition state of the LAP reaction, basedin particular on the configuration and interactionsof the phosphonate group of LPA in the active siteof blLAP.14 From the superposition it is evidentthat the interactions of bestatin in the active site ofppLAP are remarkably similar to the interactions ofLPA in the active site of blLAP, notwithstandingthe significant differences between both inhibitors.In particular, the terminal amino groups of LPAand bestatin are bound at equivalent positions andmake identical interactions in the active site ofblLAP and ppLAP, respectively, while the C2hydroxyl group of bestatin binds at the samemetal-bridging position as one of the threephosphoryl oxygens of LPA (O1 in Fig. 5). TheP–O bond associated with this metal-bridgingoxygen atom is thought to represent the carbon–oxygen bond that is formed in the transition state

Fig. 4. A diagram of the bindingmode of bestatin in the active site ofppLAP. Hydrogen bonds with bes-tatin and metal-coordinating bondsare indicated with broken lines.

Fig. 5. Stereo diagram with a comparison of the bestatin-bound ppLAP structure with the LPA-bound blLAPstructure. Bestatin (ball and stick) and residues of ppLAP (lines) are shown in magenta, LPA (ball and stick) and residuesof blLAP are shown in green. The residue numbers of blLAP are given in brackets. The three phosphonate oxygens ofLPA are labelled 1, 2 and 3, consistent with the description in the text.

709Structure of P. putida Leucine Aminopeptidase

upon attack of the water or hydroxide ionnucleophile on the carbonyl carbon atom of thescissile peptide bond.14 One of the other twophosphoryl oxygens of LPA (O2 in Fig. 5) isproposed to represent the oxyanion of the transi-tion state (the former carbonyl oxygen of thescissile peptide bond). In the LPA-bound blLAPstructure it is coordinated to the site-1 metal ionand hydrogen bonded to Lys262 (equivalent ofLys279 in ppLAP). The third phosphoryl oxygen ofLPA (O3 in Fig. 5) is thought to represent theformer peptide nitrogen atom of the substrate. Thisoxygen is within hydrogen bonding distance fromthe backbone carbonyl oxygen of Leu360 of blLAP(equivalent to Leu377 of ppLAP). In the ppLAP–bestatin complex the peptide bond is shifted awayfrom the dimetal centre due to the extra C–Cbackbone bond present in the P1 residue of theinhibitor. Nevertheless, the carbonyl oxygen andamide nitrogen of bestatin are close (within 1 Å) tothe positions of the O2 and O3 phosphoryloxygens of LPA, making similar, albeit weaker,interactions with ppLAP and the site-1 metal ion.This is possible due to the inverted configurationat the C3 carbon of bestatin (R instead of S) thatallows a change in overall binding orientation ofthe inhibitor such that the terminal amino group,the metal-bridging hydroxyl group and the car-bonyl oxygen all bind close to the dimetal centre,while the phenylalanine side chain occupies the S1pocket. The high degree of similarity between thebestatin-binding interactions in ppLAP and theLPA-binding interactions in blLAP provides a clearframework for modelling substrates in both theground state and transition state configuration inthe active site of ppLAP (see below).

Molecular modelling of the substrate-bindingmodes in ppLAP

To examine the structural basis for the substratepreferences and high enantioselectivity of ppLAP, thebestatin-bound ppLAP structure was used as atemplate to model the binding modes of the amideforms of the L-amino acids leucine, phenylglycine,valine, isoleucine, glutamic acid and arginine (Fig. 6).Earlier it was shown that among these compounds,the leucine and phenylglycine amides are the bestppLAP substrates.7 The valine and isoleucine amides,which have an extra methyl group connected to theirCβ atom, are poor substrates, while almost noamidase activity is measured with glutamic acidamide as a substrate. No ppLAP activity data areavailable for the amide forms of L-lysine and L-arginine, but the L-arginine amide has been reportedto form a good substrate for the highly similarecLAP.10,21 The amino acid amides were modelledinto the active site of ppLAP in energeticallyfavourable conformations, under consideration ofthe crucial binding interactions implied by thecomparison of the bestatin-bound ppLAP structurewith the LPA-bound structure of blLAP and theprobable mechanism described below. The model-ling included the placement of a nucleophilic watermolecule at the position of the hydroxyl oxygen ofbestatin in the bestatin-bound ppLAP structure. Theresults clearly show how the L-amino acid amides ofleucine and phenylglycine (with the S-configurationat their chiral Cα atom)might bind to the active site ina productive mode allowing formation of the metal-coordinating bonds by their α-amino and carbonylgroups, while their Cα side chains fit snugly in thehydrophobic S1 pocket (Fig. 6a and b). In this binding

Fig. 6. Substrate modelling. Models of ppLAP bound with various amino acid amides were prepared as described inthe text. The active site region in ppLAP (as a molecular surface), the active-site metals (as spheres) and the proteinresidues (in magenta as ball and stick) that contact the P1 side chain of the substrates in the S1 sub-pocket are depicted.Also shown are bicarbonate (BCT) and the proposed nucleophilic water molecule. The modelled substrates (in yellow asball and stick) are: (a) L-leucine amide; (b) L-phenylglycine amide; (c) L-valine amide, (d) L-isoleucine amide; (e) L-glutamate amide; and (f) L-arginine amide.

710 Structure of P. putida Leucine Aminopeptidase

mode, the Cα-H proton of the amino acid amidesubstrates is in close proximity (b3 Å) to thebackbone carbonyl oxygen of residue 377, whichleaves no space for any larger substituent at that

position, explaining why ppLAP is inactive with α-methyl-substituted amino acid amides. It explainsalso the high enantioselectivity of ppLAP, as sub-strates with an R-configuration at their chiral Cα

711Structure of P. putida Leucine Aminopeptidase

atom will either be excluded from the active site dueto steric hindrance of the Cα side chain, or due tounfavourable interactions with their Cα-linkedamino and carbonyl groups. The valine and isoleu-cine amides are poor substrates because their Cγ2methyl groups are positioned unfavourably betweenthe NH amide of Gly379 and the amino group ofLys279 (distances of 3.5–4 Å; Fig. 6c and d). Inaddition, the small hydrophobic side chain of the L-valine amide does not fully occupy the S1 pocket,thus further weakening the binding interactions. Theside chains of aspartate, asparagine, glutamate orglutamine amides could optimally fill the S1 pocket(Fig. 6e), but their polar or charged head groups arenot tolerated by the hydrophobic protein environ-ment, explaining why these amides do not formsubstrates of ppLAP. On the other hand, we predictthat the L-arginine amide is indeed a putativesubstrate of ppLAP, as its side chain is long enoughto traverse the S1 pocket with its charged head groupextending away from the protein surface (Fig. 6f).

Discussion

The structural similarities of ppLAPwith blLAP andecLAP, in particular with respect to its di-metalcoordination geometry and binding mode of bestatin,confirm that these enzymes share a common catalyticmechanism. In this mechanism, which has beenanalysed extensively for blLAP,11,13,14,16 the metal-bridgingwatermolecule or hydroxide ion observed inthe active site of theunliganded structure is believed torepresent the nucleophile that will attack the scissileamide bond of the substrate. Besides having a role inpositioning and activating the nucleophile, the twoactive site metals are important for substrate bindingand transition state stabilisation. The site-2metal ion iscrucial for binding the N-terminal amino group of thesubstrate, while the site-1metal ion binds the carbonyloxygen of the scissile amide bond and stabilizes thenegative charge that develops on this atom (theoxyanion) in the presumed tetrahedral gem–diolatetransition state. The oxyanion is further stabilized byan interaction with the nearby lysine residue (Lys297in ppLAP). The bicarbonate ion is believed to act as ageneral base in this mechanism, abstracting a protonfrom the nucleophilic water molecule and transferringit to the amino-terminal group of the P1’ product aftercleavage of the peptide bond.Our results indicate that while metal-binding site 2

in the unliganded high-pH structure of ppLAPcontains a Zn2+, metal-binding site 1 is occupiedmainly by Mn2+. Since no manganese was present inthe solutions used for protein purification andcrystallization, it must have been picked up byppLAP from the cytoplasm of E. coli during proteinexpression. Assuming that the intracellular concen-tration of freeMn2+ in E. coli is similar to that of Zn2+,these findings indicate that metal-binding site 1 ofppLAP has a higher specificity for Mn2+ than forZn2+. Alternatively, during expression in E. coli theintracellular levels of Mn2+ were significantly higher

than those of Zn2+. Whether Mn2+ is the preferredmetal ion in binding site 1 of ppLAP underphysiological conditions remains to be investigated.In addition, it is unclear whether the observedincrease in activity of (Mn2+-Zn2+)-bound versus(Zn2+-Zn2+)-bound ppLAP has biological relevance.A similar activation effect of Mn2+ has been observedfor other members of the M17 LAPs, includingblLAP,2,22 and in some of these enzymes the metal-activation effect is substrate specific.23 Although theprecise mechanistic basis for these effects is unclear, itmust result from subtle differences in the active site,depending on which metal ion is present at the low-affinity site. Such a metal-dependent modulation ofactivity could serve as a regulatory mechanism toalter the hydrolytic activity of LAPs towards certainsubstrates in response to changes in the environment.However, in the case of ppLAP it can be argued thatthe Mn2+ activation is only a secondary effect, andthat the metal exchangeability of site 1 serves merelyto make the enzyme more robust and less vulnerableto large fluctuations in the environment.The present crystal structures of ppLAP and their

analysis clearly explain the pH-dependence of thisenzyme and its high enantioselectivity, and providea structural basis for its observed substrate speci-ficity. They also suggest target residues for muta-genesis (e.g. Met287, Ile382, Ala466) in order tochange the substrate specificity and thus may serveas a platform for future protein engineering toenhance the applicability of this enzyme in thestereoselective synthesis of proteinogenic and non-proteinogenic L-amino acids.

Materials and Methods

Purification and crystallization

PpLAP was produced by heterologous expression in E.coli, using the expression vector pTrpLAP as described.6

All enzyme purification steps were done at 7 °C, followinga procedure based on previously established protocols.5-7

In brief, 10 g of bacterial pellet was suspended in buffer A(20 mM Hepes–KOH, pH 8.0, 1 mM DTT) containing200 mM MgSO4, which was followed by sonication andhigh-speed centrifugation to obtain a cell-free extract. Theresulting supernatant was filtered, dilutedwith buffer A toa final concentration of 25 mM MgSO4 and subsequentlyloaded onto a 6 ml Resource-S cation-exchange column(GE Healthcare), previously equilibrated with buffer Acontaining 25 mM MgSO4. Protein was eluted by a lineargradient of 25 mM to 500 mM MgSO4. The ppLAP-containing fractions were pooled and then further purifiedon a Superdex 200 10×300 mm gel-filtration column (GEHealthcare), using buffer A containing 100mMMgSO4 as arunning buffer. ppLAP eluted from the column as a singlepeak (apparent molecular mass 270 kDa) corresponding tothe expected molecular mass of a hexameric species.Purified ppLAP was concentrated to 8 mg/ml in bufferA, and subsequently used for crystallization. Initialscreening for crystallization conditions was done inhanging drops using different commercial screens. Subse-quent optimization resulted in the growth of X-ray

†http://www.pymol.org

712 Structure of P. putida Leucine Aminopeptidase

diffracting crystals at two different conditions, at low pH(8 mg/ml protein, 11% (w/v) PEG 8000, 0.2 M sodiumformate, 0.1 M Mes–NaOH, pH 5.2, 1 mM NaN3 at 5 °C)and at high pH (4 mg/ml protein, 15% (w/v) PEG 1500,0.1 M propionic acid, cacodylate, bis-Tris propane (PCB)cocktail buffer, pH 9.5 at 23 °C). The low-pH ppLAPcrystals (apo form) were hexagonal and reached anaverage size of 0.3 mm×0.3 mm×1.0 mm within threedays, whereas the high-pH ppLAP crystals (active form)were triclinic and grew overnight to an average size of0.1 mm×0.1 mm×0.1 mm. To obtain well diffractingcrystals of a bestatin-bound ppLAP–inhibitor complex itwas necessary to first dialyse purified ppLAP against20 mMHepes–KOH, pH 8.0, 0.1 M K2SO4, 100 mM EDTA,followed by extensive dialysis against EDTA- and metal-free buffer (treated with Chelex 100 Resin from Bio-Rad).The proteinwas then incubatedwith a sixfoldmolar excessof MnSO4 (relative to the concentration of the ppLAPhexamers) for 2 h at 23 °C, before adding a 30-fold molarexcess of bestatin. Hexagonal crystals of the ppLAP–bestatin complex (0.12 mm×0.12 mm×0.10 mm) wereobtained from 0.2 M DL-malic acid, pH 7.0, and 22.5%(w/v) PEG 3350 at 23 °C.

Data collection and structure determination

Diffraction data for the ppLAP crystals were measuredwith synchrotron radiation at the ESRF in Grenoble,France. Before data collection crystals were transferred toa cryoprotecting solution and flash-frozen in liquidnitrogen. The high-pH crystals were transferred in twosteps, first to 35% PEG 1500, then to 45% PEG 1500 in0.1 M PCB cocktail buffer, pH 9.5, and the low-pH crystalswere transferred in a single step to mother liquorcontaining 35% PEG 8000. The ppLAP–bestatin crystalswere transferred to mother liquor containing 20% (v/v)glycerol. The data were processed with Mosflm andmerged using Scala as implemented in the CCP4 softwaresuite. The relevant data statistics are given in Table 1.The high-pH ppLAP crystals diffracted to 2.20 Å

resolution and belonged to space group P1. Analysis ofthe Matthews coefficient and inspection of self-rotationPatterson maps indicated the presence of one hexamer perunit cell, with a solvent content of 49% (v/v). An initial setof phases was obtained by the molecular replacementmethod using the program MOLREP.24 Using the FFASand SCRWL servers25 a search model was constructedbased on the structure of ecLAP (PDB identifier 1GYT),which included all conserved side chains with theremaining non-alanine/glycine residues truncated at theCγ atom (a so-called mixed model). Several iterations ofmanual building using the program Coot26 were alternat-ed with maximum-likelihood refinement using the pro-gram Refmac5.27 Water molecules were added using Cootduring the last refinement cycles.The low-pH ppLAP crystal used for data collection

diffracted to 2.75 Å resolution and belonged to spacegroup P63, with two subunits per asymmetric unit (solventcontent 50% (v/v)). The crystal was twinned with atwinning fraction of 0.47 as determined in CNS.28 Thetwin-related reflection intensities were averaged to simu-late the case of perfect twinning, following the suggestionmade by Yeates.29 A starting model for refinement wasobtained by molecular replacement with the programPhaser30 with a single subunit of the high-pH ppLAPhexameric structure as a search model. Model refinementwas carried out using CNS, with protocols designed fortwinned data. Model building, and the placement of watermolecules was done with Coot.

The ppLAP crystal complexedwith bestatin diffracted to1.5 Å and belonged to space group P1 with one hexamerper unit cell. An initial structure was obtained bymolecular replacement with Phaser, using the high-pHppLAP structure as a template, which was subsequentlyoptimized by refinement and model building usingRefmac5 and Coot, respectively.The model quality was validated for all structures using

Coot and MolProbity.31 The statistics of the refinedstructures are given in Table 1.

Activity assays

Metal-dependent activity assays were carried out asdescribed32 using L-phenylglycine amide as a substrate.To prepare (Zn2+-Zn2+)-bound and (Mn2+-Zn2+)-boundforms of the enzyme, EDTA-treated ppLAP was incubatedovernight at room temperature with a 13-fold molarexcess of ZnSO4 and MnSO4, respectively. After incuba-tion the enzyme was washed and concentrated to 3.7 mg/ml in 20 mM Hepes-KOH, pH 8.0, 0.1 M K2SO4 in thepresence of a 1.4-foldmolar excess (96 μM) of either ZnSO4or MnSO4. Competitive activation/inhibition was ana-lysed by incubating these ppLAP preparates for 2 h with a17-fold molar excess of ZnSO4 or MnSO4, before measur-ing the activity.

Modelling

Amide forms of the L-amino acids were modelledmanually in the active site of ppLAP using the programCoot. Coordinates and topology files of the L-amino acidamides were generated using the PRODRG2 server.33 Thebestatin-bound structure of ppLAP was used as a proteinmodel. Manual dockingwas guided by the superposition ofbestatin-bound ppLAP to L-leucinephosphonic acid-boundblLAP, restraining the positions of the N-terminal aminonitrogen, the carbonyl oxygen and the amide nitrogen to theequivalent atoms in L-leucinephosphonic acid. A watermolecule was placed at the position of the hydroxyl oxygeninbestatin, andbad contactswere removedby several cyclesof energy minimization using CNS.

Figures

Figures 1, 2, 5 and 6 were prepared using the programPyMOL†.

Protein Data Bank accession number

Coordinates and structure factors have been depositedin the Protein Data Bank under accession numbers 3H8E(unliganded, low pH), 3H8F (unliganded, high pH) and3H8G (bestatin-bound complex).

Acknowledgements

We acknowledge the European Synchrotron Ra-diation Facility for provision of synchrotron

713Structure of P. putida Leucine Aminopeptidase

radiation facilities and we thank the MX beamlinescientists for assistance in beamline usage. The workof A.K. was supported by a ZonMw (the Medicaland Health Research Council of the Netherlands)research grant.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2010.03.042

References

1. Taylor, A. (1993). Aminopeptidases: structure andfunction. FASEB J. 7, 290–298.

2. Lowther, W. T. & Matthews, B. W. (2002). Metalloami-nopeptidases: common functional themes in disparatestructural surroundings. Chem. Rev. 102, 4581–4608.

3. Bordusa, F. (2002). Proteases in organic synthesis.Chem. Rev. 102, 4817–4868.

4. Sanz, Y. (2007). Aminopeptidases. In Industrialenzymes: Structure, Function and Applications (Polaina,J. & MacCabe, A. P., eds), pp. 243–262, Springer,New York.

5. Sonke, T., Ernste, S., Tandler, R. F., Kaptein, B.,Peeters, W. P., van Assema, F. B. et al. (2005). L-selective amidase with extremely broad substratespecificity from Ochrobactrum anthropi NCIMB 40321.Appl. Environ. Microbiol. 71, 7961–7973.

6. Sonke, T., Kaptein, B., Boesten, W. H., Broxterman,Q. B., Kamphuis, J., Formaggio, J. et al. (2000).Aminoamidase-catalyzed preparation and furthertransformations of enantiopure α-hydrogen- andα,α-disubstituted α-amino acids. In StereoselectiveBiocatalysis (Pate, R. N., ed.), pp. 23–58, CRC Press,New York.

7. Hermes, H. F., Sonke, T., Peters, P. J., van Balken, J. A.,Kamphuis, J., Dijkhuizen, L. & Meijer, E. M. (1993).Purification and characterization of an L-aminopepti-dase from Pseudomonas putida ATCC 12633. Appl.Environ. Microbiol. 59, 4330–4334.

8. Kamphuis, J., Meijer, E. M., Boesten, W. H., Sonke, T.,van den Tweel, W. J. & Schoemaker, H. E. (1992).New developments in the synthesis of natural andunnatural amino acids. Ann. N. Y. Acad. Sci. 672,510–527.

9. Rawlings, N. D., Morton, F. R. & Barrett, A. J. (2006).MEROPS: the peptidase database. Nucleic Acids Res.34, D270–D272.

10. Matsui, M., Fowler, J. H. & Walling, L. L. (2006).Leucine aminopeptidases: diversity in structure andfunction. Biol. Chem. 387, 1535–1544.

11. Burley, S. K., David, P. R. & Lipscomb, W. N. (1991).Leucine aminopeptidase: bestatin inhibition and amodel for enzyme-catalyzed peptide hydrolysis. Proc.Natl Acad. Sci. USA, 88, 6916–6920.

12. Kim, H. & Lipscomb,W. N. (1993). Differentiation andidentification of the two catalytic metal binding sitesin bovine lens leucine aminopeptidase by X-raycrystallography. Proc. Natl Acad. Sci. USA, 90,5006–5010.

13. Sträter, N. & Lipscomb, W. N. (1995). Two-metalion mechanism of bovine lens leucine aminopepti-dase: active site solvent structure and binding modeof L-leucinal, a gem-diolate transition state ana-

logue, by X-ray crystallography. Biochemistry, 34,14792–14800.

14. Sträter, N. & Lipscomb, W. N. (1995). Transition stateanalogue L-leucinephosphonic acid bound to bovinelens leucine aminopeptidase: X-ray structure at 1.65 Åresolution in a new crystal form. Biochemistry, 34,9200–9210.

15. Sträter, N., Sherratt, D. J. & Colloms, S. D. (1999).X-ray structure of aminopeptidase A from Escher-ichia coli and a model for the nucleoprotein complexin Xer site-specific recombination. EMBO J. 18,4513–4522.

16. Sträter,N., Sun, L., Kantrowitz, E. R.&Lipscomb,W.N.(1999). A bicarbonate ion as a general base in themechanism of peptide hydrolysis by dizinc leucineaminopeptidase. Proc. Natl Acad. Sci. USA, 96,11151–11155.

17. Allen, M. P., Yamada, A. H. & Carpenter, F. H. (1983).Kinetic parameters of metal-substituted leucine ami-nopeptidase from bovine lens. Biochemistry, 22,3778–3783.

18. Burley, S. K., David, P. R., Sweet, R. M., Taylor, A.& Lipscomb, W. N. (1992). Structure determinationand refinement of bovine lens leucine aminopepti-dase and its complex with bestatin. J. Mol. Biol. 224,113–140.

19. Kraft, M., Schleberger, C.,Weckesser, J. & Schulz, G. E.(2006). Binding structure of the leucine aminopep-tidase inhibitor microginin FR1. FEBS Lett. 580,6943–6947.

20. Schechter, I. & Berger, A. (1967). On the size of theactive site in proteases. I. Papain. Biochem. Biophys.Res. Commun. 27, 157–162.

21. Gu, Y. Q. & Walling, L. L. (2002). Identification ofresidues critical for activity of the wound-inducedleucine aminopeptidase (LAP-A) of tomato. Eur. J.Biochem. 269, 1630–1640.

22. Carpenter, F. H. & Vahl, J. M. (1973). Leucineaminopeptidase (Bovine lens). Mechanism of activa-tion by Mg2+ and Mn2+ of the zinc metalloenzyme,amino acid composition, and sulfhydryl content.J. Biol. Chem. 248, 294–304.

23. Cappiello, M., Alterio, V., Amodeo, P., Del Corso, A.,Scaloni, A., Pedone, C. et al. (2006). Metal ionsubstitution in the catalytic site greatly affects thebinding of sulfhydryl-containing compounds to leucylaminopeptidase. Biochemistry, 45, 3226–3234.

24. Vagin, A. & Teplyakov, A. (1997). MOLREP: anautomated program for molecular replacement.J. Appl. Crystallogr. 30, 1022–1025.

25. Jaroszewski, L., Rychlewski, L., Li, Z., Li, W. &Godzik, A. (2005). FFAS03: a server for profile-profile sequence alignments. Nucleic Acids Res. 33,W284–W288.

26. Emsley, P. & Cowtan, K. (2004). Coot: model-buildingtools for molecular graphics. Acta Crystallogr. D, 60,2126–2132.

27. Murshudov, G. N., Vagin, A. A. & Dodson, E. J.(1997). Refinement of macromolecular structures bythe maximum-likelihood method.. Acta Crystallogr. D,53, 240–255.

28. Brünger, A. T., Adams, P. D., Clore, G. M., DeLano,W. L., Gros, P., Grosse-Kunstleve, R. W. et al. (1998).Crystallography & NMR system: a new software suitefor macromolecular structure determination. ActaCrystallogr. D, 54, 905–921.

29. Yeates, T. O. (1997). Detecting and overcoming crystaltwinning. Methods Enzymol. 276, 344–358.

30. McCoy, A. J. (2007). Solving structures of protein

714 Structure of P. putida Leucine Aminopeptidase

complexes by molecular replacement with Phaser.Acta Crystallogr. D, 63, 32–41.

31. Davis, I. W., Leaver-Fay, A., Chen, V. B., Block, J. N.,Kapral, G. J., Wang, X. et al. (2007). MolProbity:all-atom contacts and structure validation for proteinsand nucleic acids. Nucleic Acids Res. 35, W375–W383.

32. Duchateau, A. L., Hillemans-Crombach, M. G., van

Duijnhoven, A., Reiss, R. & Sonke, T. (2004). Acolorimetric method for determination of aminoamidase activity. Anal. Biochem. 330, 362–364.

33. Schüttelkopf, A. W. & van Aalten, D. M. (2004).PRODRG: a tool for high-throughput crystallographyof protein-ligand complexes. Acta Crystallogr. D, 60,1355–1363.