High-resolution Crystal Structure of AKR11C1 from Bacillus halodurans: An NADPH-dependent...

doi:10.1016/j.jmb.2005.09.067 J. Mol. Biol. (2005) 354, 304–316

High-resolution Crystal Structure of AKR11C1from Bacillus halodurans: An NADPH-dependent4-Hydroxy-2,3-trans-nonenal Reductase

Tobias Marquardt1, Dirk Kostrewa1, Rajkumar Balakrishnan2

Antonietta Gasperina1, Christian Kambach1, Alberto Podjarny3

Fritz K. Winkler1, Ganesaratnam K. Balendiran2* and Xiao-Dan Li1*

1Biomolecular Research, PaulScherrer Institut, 5232 VilligenSwitzerland

2Division of ImmunologyBeckman Research Institute ofthe City of Hope, 1450 E, DuarteRoad, Duarte, CA 91010, USA

3Laboratoire de Genomique etde Biologie Structurale ,IGBMC , UMR 7104 ,CNRS/INSERM/ULP , 1 rueLaurent Fries , 67404 IllkirchFrance

0022-2836/$ - see front matter q 2005 E

Abbreviations used: AKR, aldo-k4-hydroxy-2,3-trans-nonenal; r.m.s.ddeviation.

E-mail addresses of the [email protected]; [email protected]

Aldo-keto reductase AKR11C1 from Bacillus halodurans, a new member ofaldo-keto reductase (AKR) family 11, has been characterized structurallyand biochemically. The structures of the apo and NADPH bound form ofAKR11C1 have been solved to 1.25 A and 1.3 A resolution, respectively.AKR11C1 possesses a novel non-aromatic stacking interaction of anarginine residue with the cofactor, which may favor release of the oxidizedcofactor. Our biochemical studies have revealed an NADPH-dependentactivity of AKR11C1 with 4-hydroxy-2,3-trans-nonenal (HNE). HNE is acytotoxic lipid peroxidation product, and detoxification in alkaliphilicbacteria, such as B. halodurans, plays a crucial role in survival. AKR11C1could thus be part of the detoxification system, which ensures the wellbeing of the microorganism. The very poor activity of AKR11C1 onstandard, small substrates such as benzaldehyde or DL-glyeraldehyde isconsistent with the observed, very open active site lacking a binding pocketfor these substrates. In contrast, modeling of HNE with its aldehydefunction suitably positioned in the active site suggests that its elongatedhydrophobic tail occupies a groove defined by hydrophobic side-chains.Multiple sequence alignment of AKR11C1 with the highly homologous iolSand YqkF proteins shows a high level of conservation in this putativesubstrate-binding site. We suggest that AKR11C1 is the first structurallycharacterized member of a new class of AKRs with specificity for substrateswith long aliphatic tails.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: aldo-keto reductase; AKR11C1; Bacillus halodurans; HNE; lipidperoxidation detoxification
*Corresponding author
Introduction

The large superfamily of aldo-keto reductases(AKR) is presently subdivided into 15 families withmore than 120 AKRs, which occur in vertebrates,plants, yeast, eubacteria, and archaebacteria.1

Although some AKRs are functionally and structu-rally well characterized, being involved in steroid,sugar, and vitamin metabolism,2,3,4 the physio-logical role of many is unknown. AKRs are typically

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eto reductase; HNE,., root-mean-square

ding authors:

monomeric proteins of about 35 kDa, but dimericand tetrameric members do occur. All possess aconserved (b/a)8 TIM barrel fold, a number ofconserved active site residues, and they predomi-nantly use NADPH as a hydride donor. Thereaction is catalyzed by a tetrad of conservedresidues, consisting of tyrosine, histidine, aspartate,and lysine. The lysine residue, shown to be crucialfor AKR activity,5 forms a salt-bridge to thecarboxylate group of the aspartate residue andmakes a hydrogen bond to the tyrosine hydroxylgroup. It is thought that this interaction makes thetyrosine more acidic, such that it donates a protonto the histidine. This proton is thought to betransferred to the substrate together withthe 4-pro-R hydride of NADPH.6 While the overall

d.

AKR11C1 Structure and Function 305

fold is well conserved in all AKRs, remarkablealterations occur in the C-terminal region, which isimportant in providing substrate specificity.7 Inhibi-tor studies of aldose reductase (AKR1B1) showedthat the specificity pocket is not very rigid, but canadapt to various substrates/inhibitors throughconformational changes.8

To date, five bacterial AKR structures have beensolved: AKR5C from Corynebacterium,9 AKR11A/Bfrom Bacillus subtilis,10 and the Escherichia coli Tasand Ydhf proteins (S. Jeudy et al., unpublishedresults).11 Extensions of loop 3, and helices H1 andH2 could be identified as common bacterialcharacteristics, which distinguish them from theprototype eukaryotic AKRs, such as aldosereductase. In AKR5C, the first cofactor-inducedassembly of an AKR active site has been reported.4

Active site residues and residues involved incofactor binding undergo coordinated confor-mational changes that result in the active holoform. The conformational changes include residuemovements up to 8 A and substantial rearrange-ment of the loop 2 region.

AKR family 11 so far consists of the structurallycharacterized AKR11A and AKR11B from B. sub-tilis.10 AKR11A is the product of the iolS gene,which is cotranscribed with the inositol (iol) operonrepressor iolR gene. The iol operons iolRS and iolA-Jhave been shown to be involved in B. subtilisinositol catabolism.12 AKR11B is the product ofthe yhdN gene, a member of the large group of sB-dependent general stress genes induced by heat,salt, or ethanol stress.13 Both proteins resemble thecommon AKR architecture, but especially AKR11Apossesses distinct structural features and intrinsic-ally low enzymatic activity.

Gene BH1011 of alkaliphilic Bacillus halodurans14

encodes a protein with a high level of identity tovarious annotated oxido-reductases of Bacillusspecies B. clausii15 (also alkaliphilic), B. subtilis,16

B. licheniformis,17 B. anthracis,18 and B. cereus.19

Levels of identity between 64% and 70% areobserved to a non-characterized oxido-reductaseencoded by gene ABC2606 of B. clausii, the YqkFproteins of B. subtilis and B. licheniformis, and to theiolS proteins of B. anthracis and B. cereus. The yqkFgene encodes for a putative AKR of unknownfunction. Experiments with B. subtilis showed thatYqkF might be a substrate of ClpXP protease, as itaccumulates in clpX and clpP mutants.20 Aftersequence submission to the AKR superfamilyhomepage†, BH1011 has been assigned to theAKR family 11 as AKR11C1.1

Here, we present the apo and holo structuresof AKR11C1 from B. halodurans, and subsequentmodeling studies with putative substrates.AKR11C1 possesses a new stacking interactionand positive charge adjacent to the cofactor, whichfavors NADPC release. Comparative analysis ofthe apo and holo form of AKR11C1 suggests a

† http://www.med.upenn.edu/akr/

cofactor-facilitated assembly of the active site. Ourbiochemical studies have revealed high AKR11C1activity on the cytotoxic peroxidation product4-hydroxy-2,3-trans-nonenal (HNE), but only pooractivity with small AKR standard substrates.AKR11C1 could be part of an HNE detoxificationsystem, which ensures the well-being of themicroorganism. Using modeling, we have verifiedthat HNE is a possible substrate for AKR11C1, andwe suggest that AKR11C1, iolS and YqkF belong to anew class of AKRs with specificity for substrateswith long aliphatic tails.

Results

Structure determination

The crystal structure of AKR11C1 has beendetermined to 1.25 A resolution using singleisomorphous replacement with anomalous scatter-ing (SIRAS) using a thiomersal derivative of the apoform (Table 1). The structure of the holo form wasobtained by soaking with NADPH. The proteincrystallized in space group P212121 with twomonomers (referred to as monomer A and mono-mer B, respectively) per asymmetric unit. There isno strong interface between the A and B monomers,consistent with the fact that the protein is amonomer in solution. In monomer A, loop 3residues 78–92 and 78–87 are disordered in theapo and holo form, respectively. For monomer B, all297 residues of the protein are well ordered. Afterrefinement, the crystallographic R-factors of the apoand holo forms were 17.2% and 15.3%, respectively,with free R-factors of 20.6% and 18.5%, respectively.Analysis of the Ramachandran plots shows thatabout 92% of the residues are in the most favoredregions and none is in a disallowed region. Thecrystallographic information is summarized inTable 1.

Overall fold

All structurally known members of the AKRsuperfamily show a (b/a)8 TIM barrel fold with twoadditional helices, H1 and H2, located outside thebarrel in the C-terminal part. The eight repeatingstructural elements, each comprising a b-strand, aconnecting loop and an a-helix, are numbered 1through 8. H1 precedes a7 and H2 follows a8.AKR11C1 shares this conserved fold with the otherAKRs (Figure 1). Like B. subtilis AKR11A/B and theE. coli Tas protein,10,11 AKR11C1 possesses anextended loop 3 (17 residues) and a short loop 4(eight residues). A similarly long loop 3 has beenseen only in the Tas protein, with a length of 19residues. Loop 3 contains a b-hairpin motif, aspresent also in AKR11A, AKR11B, rat potassiumchannel b-subunit AKR6A2,21,22 and rat liveraflatoxin dialdehyde reductase AKR7A1.23 Theelongation of helices H1 and H2 observed with

http://www.med.upenn.edu/akr/

Table 1. Data collection, crystallographic, and refinement statistics

Apo AKR11C1

Nativea Derivative Holo AKR11C1 native

Wavelength (A) 0.97784 1.5418 0.979341.37830

Resolution (A) 38.7–1.25 45–1.9 38.7–1.3(highest shell) (1.3–1.25) (2.0–1.9) (1.4–1.3)Reflections (observed/unique) 1025,135b/178,042 506,971/103,850 913,956b/169,631Completeness (%) 94.4 (83.4) 99.8 (99.9) 99.4 (99.0)I/s 10.0 (2.7) 16.7 (4.6) 9.4 (3.5)Rsym

c 0.087 0.064 0.085Space group P212121 P212121 P212121

Unit cell parameters

a (A) 74.67 74.23 75.12

b (A) 86.57 86.22 87.54

c (A) 105.43 104.68 105.43Monomers per asymmetric unit 2 2 2Matthews coefficient 2.5 2.3 2.4Phasing powerd

Acentric (isomorph./anormal.) 0.613/0.539Centric 0.580Figure of merit (acentric/centric) 0.080/0.099Rcryst

e (%) 17.2 – 15.3Rfree

f (%) 20.6 – 18.5Wilson temperature factors (A2) 19.1 20.6r.m.s.d.g bond lenght (A) 0.016 0.015r.m.s.d. bond angles (deg.) 1.691 1.715Protein atoms 4692 4736NADPH atoms – 48Water molecules 586 683Glycerol molecules 1 3Sodium ions 1 1Sucrose molecules 1 1Sulfate ions 12 11Ramachandran plotMost-favored (%) 93.0 91.5Allowed (%) 7.0 8.5

a Merged data set.b Both Friedel mates are used in the refinement.c Rsym Z

Ph

PijIiðhÞKhIðhÞij

Ph

PiIiðhÞ, where Ii(h) and ‘I(h)’ are the ith and mean measurement of the intensity of reflection h.

d Phasing powerZSFcalcH jSjFobs

PH KFcalcPH , where Fcalc

PH is the calculated heavy atom amplitude and FobsPH and Fcalc

PH are the observed andcalculated heavy atom derivative structure factor amplitudes, respectively.

e Rcryst Z jSjFobsP KFcalc

P jSFobsP , where Fobs

PH and FcalcPH are the observed and calculated structure factor amplitudes, respectively.

f 5% test set.g Root mean square deviation from the parameter set for ideal stereochemistry.60

306 AKR11C1 Structure and Function

other bacterial AKRs is not observed withAKR11C1.

The C terminus extends over the top of the barrelin all structurally known active AKRs, and itsamino acid residues contribute to the substrate-binding site. In contrast, AKR11A and AKR6A2,which possess a C-terminal loop too short to spanthe barrel, show no or only poor catalytic activity.Although the C terminus of AKR11C1 would belong enough to span the barrel, it is anchored at thecircumference by direct and water-mediated inter-actions to a5, and to loops 5 and 6. Additionally, itsTyr292 is buried in a hydrophobic pocket formed byIle151, Pro153, Met170, Arg178, Pro179 and Trp182.It is noteworthy that the C termini of monomer Aand B can be superimposed perfectly, despitedifferent crystallographic environments indicatingthat this is likely to be the predominant confor-mation in solution and not a crystal-packingartefact. Multiple sequence alignment with highly

homologous Bacillus proteins (Figure 7)24–27 showsthat residues involved in maintaining thisC-terminal loop conformation are conserved, indi-cating that this is a common structural feature ofthese proteins.

Comparison of apo and holo enzyme

The structures of the two AKR11C1 monomers,A and B, observed in the asymmetric unit differremarkably in the active site region. That ofmonomer B is very similar to the familiar, highlyconserved active site architecture of AKRs. Soakingwith NADPH leads to cofactor binding andaccompanying structural changes, but only in thecase of monomer B. In contrast, monomer A, whichappears to be present in a catalytically unproduc-tive conformation, remains essentially unchanged.

The Ca atoms of the apo and holo form ofmonomer B can be superimposed with an r.m.s.d.

Figure 1. Stereo view of the overall fold of holo AKR11C1 (monomer B). Loop 1 (red) does not directly interact withloop 7 (orange) closing above the bound cofactor NADPH. The C terminus (yellow), although long enough, does notspan back to the active site and therefore does not contribute to forming a substrate-binding site. The C-terminal loopconformation is well maintained by H-bonds, electrostatic and hydrophobic interactions. As seen in other microbialAKRs, loop 3 (blue) instead of loop 4 is extended. It contains an additional b-hairpin motif and is involved in substratebinding/recognition. The additional helices H1 and H2 are not elongated.

Figure 2. (a) Superposition of monomer A and NADPH-bound monomer B. Regions that show significant differencesin side-chain and main-chain conformations and rigid-body movements are highlighted in pink (monomer A) andgreen (monomer B). (b) Comparison of loop 1 and loop 2 conformations of apo monomer A (pink) and holo monomer B(green). In monomer A, the catalytic site is disrupted, with Tyr51 moved away from the other catalytic tetrad residues.Steric hindering through Tyr51 and Leu50 (not shown) inhibits cofactor binding. Due to the different conformation ofloop 2, helix a2 is shorter in monomer A.


Figure 3. Close-up view of holo AKR11C1 active site. The conserved residues Asp46, Tyr51, Lys76 and His119 form ahydrogen bond/salt-bridge network. A water molecule is observed between Tyr51 and His119 at a position also reportedfor the O33 atom of the aldose reductase inhibitor IDD594. It presumably mimics the oxygen position of the substrate.The 2mFoKDFc electron density map is contoured at 0.376 e/A3 (equivalent to 1 s(e) distances in A).


value of 0.23 A. Conformational changes occur insome side-chains that are involved directly incofactor binding. Arg201 and Ser258 reorient toform H-bonds with phosphate NP and 2 0-phos-phate AP2*, respectively, and Asn266 reorients toform H-bonds with the N6 and N7 of the adeninemoiety. His237 has to move out of the way to avoida clash with the bound cofactor.

Comparison of apo monomer A and apo (or holo)monomer B reveals conformational changes in loop2 (residues 48–70) and loop 4 (119–120) at the activesite and in loop 1 (14–39), loop 7 (198–227) and loop8 (254–260), which are involved in cofactor binding(Figure 2(a)). Due to the remarkable change in itsloop 2 conformation, the catalytic site of monomerA is disrupted completely, with the catalyticallyimportant residue Tyr51 being flipped out of theactive site and buried in a pocket formed by Glu171,Arg196 and Arg201. In addition, Tyr51 is involvedin a crystal contact. Compared to its position in theholo form, Tyr51 is displaced by about 9 A. Themain chains of monomer A and B start diverging atAla48 and, after taking a completely differentcourse, come together again at the second turn ofhelix a2 of monomer B (Figure 2(b)). The extrusionof Tyr51 from the active site results in the break-down of the conserved H-bond network betweenthe catalytic residues Asp46, Tyr51, Lys76 andHis119. Instead, the reoriented side-chains ofLys76 and His119 form a new, water-mediated,network with Asp46. Due to steric clashes, the loop2 conformation of monomer A is not compatiblewith cofactor binding. Apparently, the necessarylarge structural changes are prevented by thecrystal packing.

Catalytic site

The conserved active site architecture of AKRscomprising the catalytic tetrad Asp46, Tyr51, Lys76and His119 is observed only in monomer B(Figure 3). It can be superimposed perfectly withthose of human aldose reductase,28 3aHSD,2 rat

AKR6A2,22and B. subtilis AKR11B,10 reflecting thehigh degree of conservation through AKR familiesand species. As seen in most AKRs, Tyr51 ishydrogen bonded to Lys76, which also forms aconserved salt-bridge to Asp46. A water moleculebridges the hydroxyl group of Tyr51 and N3 ofHis119. Interestingly, it occupies the same positionreported for the O33 oxygen atom of the aldosereductase inhibitor IDD594.28

Cofactor binding

The cofactor NADPH is bound in an extendedconformation, as seen in most AKRs, through atight network of hydrogen bonds (Figure 4) invol-ving residues of loops 1, 7, and 8, and helix a8. Theyform a cleft spanning the C-terminal face of thebarrel. Usually, loops 1 and 7 close above the boundcofactor and form direct interactions with eachother. As in the case of AKR11A, this is not observedfor AKR11C1 cofactor binding, and the confor-mations of loop 1 and loop 7 are unchanged uponbinding. Instead, the side-chains of Arg201 andArg207 change conformation and partly embracethe cofactor. The guanidinium group of Arg196stacks against the nicotinamide moiety of thecofactor. In all other known structures, this stackinginteraction is made by the aromatic ring of atyrosine, phenylalanine or tryptophan residue.The repulsion between the positive charge of thearginine and the oxidized cofactor NADPC mayfavor cofactor release, which is believed to be therate-determining step.29 In accordance, soakingwith NADPC was not successful.

Substrate specificity

Among several potential AKR substrates testedfor their activity (Table 2), AKR11C1 shows aremarkably high level of activity on the cytotoxiclipid peroxidation product HNE (Figure 5) with aKm of 48 mM and a turnover rate, kcat, of 9.5 minK1.The inhibitor S-(4-nitrobenzyl)glutathione showed

Figure 4. Schematic of interactions between the cofactorNADPH and AKR11C1. NADPH is bound to the enzymethrough a tight network of H-bonds to side-chain andmain-chain atoms, and water molecules. In contrast to allother structurally known AKRs, where the nicotinamidemoiety stacks against an aromatic side-chain, inAKR11C1, the nicotinamide moiety stacks against thepositively charged guanidinium group of Arg196.

Table 2. Enzyme kinetic parameters of the compoundstested for substrate specificity of AKR11C1

Substrate Km kcat (minK1)

HNE 48 mM 9.5DL-glyceraldehyde 187 mM 0.4Benzaldehyde 1.7 mM 3.5p-Nitrobenzaldehyde 1.2 mM 2.5Methylglyoxal 0.86 mM 3.4Butyraldehyde 1.9 mM 2.2Pyridoxal – –D-arabinose – –L-xylose – –


a non-competitive mixed pattern of inhibition inthe presence of HNE as the substrate with a Kii of70.2(C16.3) mM and Kis of 1.95(C2.1) mM.

AKR11C1 can reduce the common AKR sub-strates DL-glyceraldehyde, benzaldehyde, p-nitro-benzaldehyde, methylglyoxal, and butyraldehydein an NADPH-dependent manner, but with ratherlow turnover rates that are between 0.4 minK1 and3.5 minK1 (Table 2). For other AKRs, much largerturnover rates in the range of 61 (human aldose

reductase, DL-glyceraldehydes)5 to 320 minK1

(AKR11B, methylglyoxal)10 have been reported.Compared to the values observed for AKR11C1with the substrates listed in Table 2, human aldosereductase, B. subtilis AKR11A/B, or E. coliAKR14A1 are from 44-fold to O300-fold moreactive.5,10,30 Because of the high level of sequenceidentity (61%) with a B. cereus D-threo-aldose1-dehydrogenase,19 and with the recently identifiedpyridoxal 4-dehydrogenase AKR15A1 (26%),31,32

we tested whether AKR11C1 can catalyze theoxidation of pyridoxal, L-xylose and D-arabinoseusing NADPC as the hydride acceptor. However,no activity was detectable, suggesting thatAKR11C1 does not act as a dehydrogenase.

Substrate binding and recognition

All structurally characterized and catalyticallyactive AKRs possess a C-terminal loop bridgingover the top of the barrel to the active site. Togetherwith other residues, the C-terminal loop residuesform the substrate-binding pocket. In AKR11C1, theC-terminal loop does not span back over the top ofthe barrel and no obvious substrate-binding pocketcan be identified. The active site is thus very openand located at the end of the hydrophobic cofactor-binding cleft. To address the question of why HNEis a much better substrate than the smaller AKRstandard substrates benzaldehyde and DL-glycer-aldehyde, we modeled HNE, benzaldehyde, andDL-glyceraldehyde in the presumed active site ofAKR11C1, using the position of the active site-bound water molecule (position of the O33 oxygenatom of IDD594) as docking site for the aldehydeoxygen atom (Figure 6(a)). The modeled HNEmolecule (using the energy-minimizing procedureand default weights of MOLOC,33 coordinates ofAKR11C1 and NADPH kept fixed) can interactfavorably with a shallow hydrophobic groovedefined by the side-chain of Trp88 (Figure 6(b)).The aldehyde group of HNE can form hydrogenbonds to Tyr51 and His119, and Met21 interactswith the hydroxyl group of the substrate. Incontrast, the benzene ring of a benzaldehyde-likemolecule or the hydroxyl groups of DL-glyceralde-hyde cannot be oriented suitably to interact with theprotein surface.

Figure 5. HNE structure andformation. HNE arises from ara-chidonic acid after reaction withreactive oxygen species (ROS).


Multiple sequence alignment of AKR11C1 withthe highly homologous iolS and YqkF proteins, andB. clausii ABC2606 (Figure 7) shows that Met21,Leu50, and Trp88, which shape the hydrophobicgroove, are conserved. Loop 3, which is adjacent tothe predicted substrate-binding groove, is wellconserved with aromatic residues Phe81 (Trp81 iniolS and YqkF) and Trp88, which are hydro-phobically buried in AKR11C1. The less conservedresidues 82–86 are solvent-exposed.

Discussion

Sequence analysis of the protein encoded by geneBH1011 of B. halodurans has classified it asAKR11C1, a member of family 11 of the aldo-ketoreductase superfamily. Its structure analysis showsthat it indeed shares the highly conserved stereo-chemistry of the active site observed for manywell-characterized members of this superfamily.Furthermore, the observed binding of NADPH butnot NADPC in crystal soaking experimentssuggests that this protein functions as a reductase.AKR11C1 is the first AKR that has been shown tohave a positively charged guanidinium group(Arg196) stacked on the nicotinamide moiety ofthe cofactor. In all other AKR structures, anaromatic stacking with the side-chain of a tyrosine,phenylalanine, or tryptophan residue is observed.Release of the oxidized cofactor is believed to be therate-determining step of AKRs,29 and chargerepulsion possibly facilitates its dissociation inAKR11C1. In contrast to other AKRs, no directinteraction of loop 1 and loop 7 (“safety belt”)34

with the bound cofactor is observed in AKR11C1nor do these loops change conformation uponcofactor binding. In aldose reductase, for example,a tight loop closure over the nucleotide has beenshown to be crucial for optimal enzyme activity.35

The absence of a safety belt-type interaction withNADPH and the presence of the nicotinamideArg196 stacking interaction are distinctive featuresof AKR11C1 cofactor binding.

We observe two very distinct active site confor-mations in AKR11C1 crystals. Monomer B pos-sesses the highly conserved active site architectureof AKRs. In contrast, remarkable conformationalchanges in monomer A result in a completelydisrupted catalytic site. The catalytically importantresidue Tyr51 is flipped out of the active site, buriedin a hydrophobic pocket, and is additionallyinvolved in a crystal contact. Due to steric clashes,monomer A is not compatible with cofactorbinding. Sanli and Blaber have reported a cofactor-induced assembly of the active site of AKR5C4 bycomparison of their apo and holo AKR5C struc-tures. Interestingly, in the two different crystalforms of AKR5C, the homologous residue Tyr50 issimilarly involved in a crystal contact in the apoform. However, we do not know whether theunproductive conformation of AKR11C1 (monomerA) exists also in solution or is a crystal packingartefact. Consequently, we do not know if it hasfunctional significance.

The biological function of AKR11C1 is unknown,but we have been able to demonstrate a convincingenzymatic activity with HNE, in contrast tostandard substrates. The affinity of AKR11C1 forHNE is comparable to those of hepatic and otherglutathione-S-transferases for HNE, which arebetween 50 mM and 100 mM,36–38 and slightlylower than those of aldose reductase andAKR1C1, which are 31 mM and 34 mM, respect-ively.39,40 Our results show no substrate-inducedinactivation of either apo or holo AKR11C1 duringcatalytic reduction of HNE. In contrast, cardiacaldose reductase41 and human placental aldosereductase (G.K.B., unpublished results) apoenzymes were found to be inactivated by HNE,

Figure 6. Modeled enzyme–substrate interactions.(a) Models of HNE (green), benzaldehyde (blue) andDL-glyceraldehyde (yellow) at the AKR11C1 active site.See the text for details. (b) Surface presentation of HNEbound to AKR11C1. The substrate fits in the shallowgroove defined by Trp88 and Met21.


while the NADPH binary complex was not affected.HNE reacts avidly with thiol groups,42 and couldpotentially inactivate aldose reductase, whichcontains a highly reactive thiol group (Cys298) atthe active site. It is very sensitive to oxidation,43 andhas been suggested to play a regulative role.44 Inline with this hypothesis, AKR11C1, which lackssuch a reactive cysteine residue, shows unalteredactivity with HNE after preincubation in theabsence of NADPH.

So far, only three AKR structures with boundsubstrate or substrate-like molecules (aldosereductase with glucose 6-phosphate, 3aHSD withtestosterone) have been solved.2,45,46 3aHSD isactive on steroid substrates and has an extended,

predominantly hydrophobic, substrate-bindingpocket.2 Aldose reductase possesses an anion-binding site within the active site pocket.46 In boththese cases,2,28 and in other active AKRs, theC-terminal loop spans back over the barrel andsome of its residues provide the interaction surfacefor bound substrate or inhibitor molecules. This isnot the case with AKR11C1, whose C-terminal loop,although long enough to span the barrel, isanchored stably on the protein surface somedistance away from the active site. Our substratemodeling studies reveal that the benzene ring of abenzaldehyde-like molecule cannot interact favor-ably with the protein surface in allowed confor-mations, and cannot be expected to be a goodsubstrate. This is the case also for DL-glyceralde-hyde. In contrast, the alkyl chain of HNE, with itsaldehyde function positioned suitably in the activesite, can be fit smoothly into a shallow, hydrophobicsurface groove in a strain-free conformation. Theresidues and regions (loops 1, 2, and 3) shown to beinvolved in HNE binding are highly conserved inABC2606, and in the iolS and YqkF proteins, whosesubstrates are unknown. Based on the high level ofconservation in the putative substrate-bindingregion, in the distinctive anchoring of theC-terminal tail and in the NADPH-bindingmode, we suggest that AKR11C1, ABC2606, andthe iolS and YqkF proteins belong to a new class ofAKRs that act on substrates with long aliphatic tailssuch as lipids, fatty acids or their degradationproducts.

The extreme living environment of B. halodurans,with growth under alkaline conditions,14,47 requiresefficient detoxification of lipid peroxidationproducts. It is known that in the cytoplasmicmembrane of alkaliphilic bacteria, polyunsaturatedfatty acids predominate over saturated fatty acids.48

Reactive oxygen species (ROS) interact readily withpolyunsaturated fatty acids in membranes andform cytotoxic aldehydes such as HNE andmalondialdehyde,42 which, in turn, are capable ofinactivating membrane proteins and thus can bedetrimental to the survival of alkaliphilic bacteria.Especially in alkaliphiles, the cytoplasmic mem-brane has a crucial function in maintaining optimalinternal conditions for metabolism and energytransduction, as the bacterium has to maintain anoptimal internal pH of 8, in contrast to an externalpH O9.5, through NaC/HC antiporter systems.47,49

AKR11C1 could function in this manner, being partof a detoxification system that ensures the function-ality of membrane proteins, such as the NaC/HC

antiporter, and hence the survival of the microor-ganism. The presence of highly homologous proteinABC2606 in another alkaliphilic (B. clausii) strength-ens this supposition. However, analysis of thegenomic neighborhood of AKR11C114 shows thatit is part of a gene cluster of unknown conservedproteins and cannot be linked to functional knownoperons or metabolisms, respectively. Furtherstudies will be necessary to elucidate and validatethe function of AKR11C1 in vivo.

Figure 7. Multiple sequence alignment of AKR11C1 with Bacillus sp. YqkF, iolS, and ABC2606. Conserved catalyticresidues and residues involved in C-terminal loop conformation, cofactor-binding and substrate-binding are markedwith a triangle, circle, square, or cross, respectively. The secondary structural elements of AKR11C1 are marked abovethe sequence. Sequences are: AKR11C1 from B. halodurans, accession no. NP_241877; B. licheniformis YqkF, accession no.AAU24034; B. subtilis YqkF, accession no. NP_390243; B. anthracis iolS, accession no. NP_846551; B. cereus iolS, accessionno. AAS43066; DTA1D, D-threo-aldose 1-dehydrogenase from B. cereus, accession no. NP_833814; ABC2606 fromB. clausii, accession no. YP_176102.


Materials and Methods

Cloning, expression, and purification

Gene BH1011 was amplified from genomic B. halo-durans strain C-125 DNA using the polymerase chainreaction (PCR) and cloned in the NdeI/XhoI sites ofexpression vector pET15b (Novagen). The protein wasproduced in E. coli strain B834 (DE3) which had beentransformed with the vector: 1 l of LB medium containing100 mg/ml of ampicillin was inoculated with overnightcolonies of transformed cells and grown at 37 8C until anabsorbance at 600 nm (A600) of about 2.5 was reached.

Cells were harvested by centrifugation, resuspended in50 mM sodium phosphate buffer (pH 8) containing300 mM NaCl, 20 mM imidazole, 10% (v/v) glyceroland sonicated at 4 8C. After centrifugation at 40,000g, thesupernatant containing the soluble protein was loadedonto a 5 ml HiTrap chelating HP (Amersham) nickelcolumn and eluted using an imidazole step gradient.Fractions containing the protein were combined, concen-trated and a buffer exchange to 20 mM Tris–HCl (pH 8),100 mM NaCl, 10% (v/v) glycerol was performed by gel-filtration chromatography using a Superdex 200 10/30column (Amersham). After gel-filtration, the yield of pureprotein was 50 mg/l of culture.


Crystallization and soaking procedures

Crystals of the apo enzyme were grown at 20 8C usingthe hanging-drop method. The protein was concentratedto 40 mg/ml and crystallized by mixing 4 ml of proteinsolution and 4 ml of well solution (1.8 M ammoniumsulfate, 0.6 M lithium sulfate, final pH 5.5) as precipi-tants/salts. Crystals appeared after four days andcontinued growing for weeks. All experiments wereperformed with crystals of sizes about 0.4 mm!0.3 mm!0.3 mm.

For phasing experiments with heavy-metal derivatives,crystals were soaked over night with 1 mM thiomersalin crystallization buffer. For obtaining the holo struc-ture, crystals were soaked over night with 10 mMNADPH.

Data collection and processing

Crystals were soaked with 35% (w/v) sucrose incrystallization buffer for cryo-protection, mounted usinga nylon-fiber loop and flash-cooled to 100 K in a nitrogenstream. Native data of the apo and holo forms werecollected as consecutive series of 0.258 rotation images atbeamline X06SA (Swiss Light Source, Villigen) on anMAR CCD detector. Derivative data were collectedin-house as consecutive series of 0.58 rotation images onan MAR345 image plate-detector using CuKa radiationproduced by an Enraf-Nonius FR591 rotating anodegenerator. Native and derivative data were indexed andintegrated with XDS,50 scaled with XSCALE, andconverted to CCP4 or SHELX format using XDSCONV.For phasing, SHELXC, SHELXD51 and SHARP52 wereused, applying the single isomorphous replacement withanomalous scattering (SIRAS) method with the nativedata as the reference data set. After solvent flattening withSOLOMON,53 we had a fully interpretable electrondensity map.

Model building and refinement

ARP/wARP54,55 was used for initial model building.ARP/wARP built a complete main chain. The correctside-chains were modeled into the electron density mapsfollowed by real space refinement using the computergraphics program MOLOC.33 Iterative rounds of modelbuilding using MOLOC and maximum-likelihood refine-ment using REFMAC5.2 completed the model. Thequality of the model was finally checked with PRO-CHECK,56 WHAT CHECK,57 and ROTAMER (against thepenultimate rotamer library).58

Enzyme kinetics

AKR11C1 activity was determined at 25 8C as the rate ofdecrease in the absorption of NADPH at 340 nm using aBeckman DU640 model spectrophotometer. The activityof the enzyme is represented as mmol of NADPH oxidizedminK1 lK1. The assay mixture (1 ml final volume) was0.2 M potassium phosphate buffer (pH 6.2), 0.15 mMNADPH, 0.5 mM AKR11C1, 0–100 mM 4-hydroxy-2,3-trans-nonenal (HNE), or 0–10 mM of one of the substratesmethylglyoxal, benzaldehyde, p-nitrobenzaldehyde,and butyraldehyde. The experimental design includedfive concentrations (0, 1 mM, 10 mM, 50 mM, and 100 mM)of the inhibitor S-(4-nitrobenzyl)glutathione with fourdifferent concentrations of the substrate, HNE (5 mM,10 mM, 50 mM, and 100 mM). Each individual rate

measurement was evaluated in duplicate. At least threeindependent determinations were performed for eachkinetic constant.

The reverse dehydrogenase assay was performed asdescribed for AKR15A1.31,32 The assay mixture (1 ml finalvolume) was 50 mM sodium phosphate buffer (pH 8),1 mM NADPC, 0.1 mM AKR11C1, and 250 mM sugarsubstrates, or 0.5 mM pyridoxal. The rate of increase inthe absorption of NADPH or 4-pyridoxolactone wasfollowed at 25 8C by measuring absorbance at 340 and366 nm, respectively, using a Shimadzu UV-1601 spectro-photometer.

Enzyme kinetics data analysis

The Michaelis-Menten constant, Km, the maximumvelocity Vmax of HNE reduction, and the inhibitionconstant, Ki for the inhibitor were calculated using thecommercial software package Prism (Graphpad), using anon-linear Marquardt’s regression algorithm.59 Theuntransformed kinetic data (mean of duplicates) werefit to a one-substrate Michaelis-Menten equation (1). Todetermine the Ki value corresponding to the inhibitor,competitive, non-competitive and uncompetitive modelsof inhibition were investigated using equations (2)–(4),respectively (where v is the rate of reaction, Vmax is themaximum initial velocity for the uninhibited reaction, I isthe inhibitor concentration, Kis is the slope inhibitionconstant and Kii is the intercept inhibition constant).The Ki values of the inhibitors were calculated from thesecondary plot of slope values of the double-reciprocalplot versus inhibitor concentration. Goodness of fitwas assessed using the r2 test. Values were expressedas the mean and standard error for three replicateexperiments.

v ZVmaxS

ðKm CSÞ(1)

v ZVmaxS

Kmð1 C l=KiÞCS(2)

v ZVmaxS

Kmð1 C l=KisÞCSð1 C l=KiiÞ(3)

v ZVmaxS

Km CSð1 C l=KiÞ(4)

Static light-scattering

For determination of the oligomeric state insolution, static light-scattering experiments wereperformed using a miniDAWN TriStar with Optilabrex refractometer (Wyatt) coupled to a Superdex 20010/30 gel-filtration column on an Agilent 1100 seriesHPLC. 0.1 mg of protein in 20 mM Tris–HCl (pH 8),100 mM NaCl was injected onto the column equili-brated with the same buffer. The mass of the nativeprotein was calculated using Wyatt Astra softwareversion 4.90.08.

Atomic coordinates and structure factors

The atomic coordinates and structure factors (codes1YNP, 1YNQ) have been deposited in the Protein


Data Bank, Research Collaboratory for StructuralBioinformatics, Rutgers University, New Brunswick,NJ†.

Acknowledgements

We thank Andrea Prota for help in initial datacollection and the illustrations, and ClemensSchulze-Briese, Ehmke Pohl, and Claude Prader-vand for help with data collection at the SLS.This work was supported by PSI ResearchCommission grant FK-05.02.1, the Swiss NationalScience Foundation, within the framework of theNational Center of Competence in ResearchStructural Biology program, by funding fromthe American Diabetes Association, and wassupported by the Centre National de laRecherche Scientifique (CNRS), by the InstitutNational de la Sante et de la Recherche Medicaleand the Hopital Universitaire de Strasbourg(H.U.S.).

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Edited by M. Guss

(Received 24 May 2005; received in revised form 20 September 2005; accepted 21 September 2005)Available online 10 October 2005

High-resolution Crystal Structure of AKR11C1 from Bacillus halodurans: An NADPH-dependent...

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