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Protein Structures: Kaleidoscope of Structural Properties and Functions, 2003: 441-443 ISBN: 81-7736-177-5

Editor: Vladimir N. Uversky

19

NAD+-dependent formate dehydrogenase. From a model enzyme to a versatile biocatalyst
Vladimir O.Popov1 and Vladimir I.Tishkov2 1 A.N.Bakh Institute of Biochemistry, Russian Academy of Sciences Leninskiy pr., 33 119071 Moscow, Russia. E-mail: vpopov@inbi.ras.ru; 2 Department of Chemical Enzymology Chemistry Faculty, M.V.Lomonosov Moscow State University Leninskie Gory, 119992 Moscow, Russia. E-mail vit@enz.chem.msu.ru

I. Introduction
Very few enzymes enjoyed such a close attention from various sectors of life sciences community ranging from quantum chemists to biotechnologists as NAD+-dependent formate dehydrogenase (EC 1.2.1.2; FDH). First discovered in 1950-1951 [1,2] it has not attracted much interest until the middle of seventies of the last century when a problem of cofactor regeneration was brought on the agenda due to the use of redox enzymes for synthesis of high value added organic chemicals. Quite soon it was realized also that FDH is one of the most suitable models for investigating the general mechanism of catalysis involving hydride ion transfer. The chemical reaction catalyzed by FDH is devoid of proton release or abstraction steps and entails cleavage of a single
C o rre s pondence/R e print re ques t: Prof. Vladimir O. Popov, La boratory of Enzyme Engineering, A. N. Ba kh Ins t it ut e of Bioche mis t ry, R us s ian Academy of Scie nces , Le nins kiy pr. , 33, 119071 M o s c ow. E-ma il: vpopov@inbi. ra s . ru


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carbon-hydrogen bond in the substrate and formation of a single new one in the product. This fortunate combination of advantageous intrinsic enzymatic and physico-chemical properties alongside with a high biotechnological potential brought FDH into the focus of research in the field of NAD+-dependent dehydrogenation. By now NAD+-dependent FDH is one of the most extensively studied among NAD+dependent dehydrogenases and can be regarded as a "text-book" enzyme along with such well-known examples as alcohol dehydrogenase or lactate dehydrogenase. State of the art in FDH research was last comprehensively reviewed in 1994 [3]. The present review provides an update to the previous one, is confined to the recent developments only and focuses on the modern understanding of the structure and mechanism of action of NAD+dependent FDH and issues relevant to its biotechnological applications.

II. Structure
Sequence
The past decade and especially several recent years due to the progress in the field of genomics resulted in the outburst of the FDH sequences form various sources. Genes of FDH were found in uncultured proteobacterium EBAC31A08 (EMBL Accession AF279106); bacteria - Pseudomonas sp. 101 [4], Moraxella C-1 (EMBL Y13245), Paracoccus sp. 12A [5], Mycobacterium vaccae N10 [6], Hyphomicrobium sp. JC17 [7], in pSymA megaplasmid of symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti [8]; methylotrophic yeast - Pichia angusta (previously named Hansenula polymorpha) [9], Candida methylica [10], Candida boidinii [11,12], baker's yeast Saccharomyces cerevisiae (EMBL Accession Z75296); fungi - Aspergillus nidulans [13], Neurospora crassa [14], Magnaporthe grisea (EMBL AA415108); higher plants ­ potato Solanum tuberosum [15], barley Hordeum vulgare [16], rice Oryza sativa (EMBL AB019533), Arabidopsis thaliana (EMBL AB023897) and even mammals ­ N-terminus of FDH from mouse Mus musculus (translated from partial cDNA, EMBL AI505623). By now more than 25 full and partial sequences of NAD+-dependent FDHs or ORFs attributed to FDH are documented compared to 4 known in 1994. Some of them representing various classes of organisms are shown in Fig. 1 (numbering of the amino acid residues throughout the paper refers to that in PseFDH). All the FDHs sequenced to date show strong similarity in primary structures. Identity within a group of organisms (bacteria, yeast, fungi, plants) is usually around 80+ %. However even the distantly related organisms such as FDHs from bacteria and higher plants reveal more than 50 % identity. A specific feature of bacterial sequences is a long N-terminal irregular loop (amino acid residues 12-46) present also in the mouse sequence but substituted by a short 6 residues segment in yeasts, fungi and plants, Fig.1. All the amino acid residues critical for catalysis or coenzyme and substrate binding are highly conserved, Fig. 1. The extent of similarity in and around the active site region is as high as 95 %, all the few substitutions compared to bacteria are for homologous amino acid residues and specific to a group of organisms (e.g. I122V substitution in yeasts or T282N in plants). A notable exception is SceFDH where two conservative prolines, Pro312 and Pro332 are substituted for lysine and valine respectively. FDH falls into the family of D-specific 2-hydroxyacid dehydrogenases acting on Dstereoisomers of respective substrates. Significant sequence similarity has been detected within this family [3,17]. Recent structural studies of D-specific dehydrogenases -


Formate dehydrogenase
PseFDH MorFDH ParFDH MmuFDH PotFDH BarFDH RicFDH SceFDH CboFDH HanFDH MagFDH NeuFDH PseFDH MorFDH ParFDH MmuFDH PotFDH BarFDH RicFDH SceFDH CboFDH HanFDH MagFDH NeuFDH PseFDH MorFDH ParFDH PotFDH BarFDH RicFDH SceFDH CboFDH HanFDH MagFDH NeuFDH LheDLD LbuDHD HmeDGD EcDPGD EfVanH PseFDH MorFDH ParFDH PotFDH BarFDH RicFDH SceFDH CboFDH HanFDH MagFDH NeuFDH PseFDH MorFDH ParFDH PotFDH BarFDH RicFDH SceFDH CboFDH HanFDH MagFDH NeuFDH ..... ..... ..... ..... ............... ............... ............... ............... 1 ..........<---------->..'.........'.........'... ..........AKVLCVLYDDPVDGYPKTYARDDLPKIDHYPGG ..........AKVVCVLYDDPINGYPTSYARDDLPRIDKYPDG ..........AKVVCVLYDDPVDGYPTSYARDSLPVIERYPDG AKILCVLYPDPVDGYPPVYARDSIPYIGGYPDG TRELQASPGPKKIVGVFYKAN------EYAEMN---------SRAAHTSAGSKKIVGVFYQAG------EYADKN---------SRAAHTSAGSKKIVGVFYKGG------EYATKN---------SKGKVLLVLYEGG------KHAEEQ---------...........KIVLVLYDAG------KHAADE---------...........KVVLVLYDAG------KHAQDE---------LTTQREKVKVLLVLYDGG------QHAKDV---------..........VKVLAVLYDGG------KHGEEV---------......'....... QTLPTPKAIDFTPG QTLPTPKAIDFTPG QTLPTPKAIDFVPG QSLATPSAIDFTPG -------------P -------------P -------------P -------------E -------------E -------------E -------------P -------------P 3/10-1A 1 ..'....<------><------------>.68 QLLGSVSGELGLRKYLESNGH ALLGSVSGELGLRKYLESQGH SLLGSVSGELGLRNYLEAQGH ELLGCVSGELGLRxYLEAQGH NFLGCAENALGIREWLESKGH NFVGCVEGALGIRDWLESKGH NFVGCVEGALGIREWLESKGH KLLGCIENELGIRNFIEEQGY KLYGCTENKLGIANWLKDQGH RLYGCTENALGIRDWLEKQGH ELLGTTENELGIRKWLEDQGH ELLGTIQNELGLRKWLEDQGH

443

.SRVASTAARAITSPSSLVF ...AAMWRAAARQLVDRAVG AMWRAAAGHLLGRALG .................... .................... .................... 4 2 <------>........<----------> TLVVTSDKDG-PDSVFEREL ELVVTSSKDG-PDSELEKHL ELVVTSSKDG-PDSELEKHL ELVVTSDKDG-PDSVFEKEL QYIVTPDKEG-PDCELEKHI HYIVTDDKEG-FNSELEKHI HYIVTDDKEG-LNSELEKHI ELVTTIDKDPEPTSTVDREL ELITTSDKEGE-TSELDKHI DVVVTSDKEGQ-NSVLEKNI TLVTTSDKDGE-NSTFDKEL TLVTTCDKDGE-NSTFDKEL 5 6 .<------------------>..<---------- NYLPSHEWARKGGWNIADCV NYIPSHDWARNGGWNIADCV NYTPSHDWAVKGGWNIADCV NFLPGHHQVINGEWNVAAIA NFLPGYQQVVKGEWNVAGIA NFLPGYQQVVHGEWNVAGIA NYNGGHQQAINGEWDIAGVA NFVPAHEQIINHDWEVAAIA NFVPAHEQIISGGWNVAEIA NFVPAHEMIQAGEWDVAGAA NFVPAHEQIQEGRWDVAEAA

3 7 4 8 A 5 ...<------>.....'..<------------>...<-------->'......<------------------>.<-->.....<-------------------------------->163 VDADVVISQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAID--RNVTVAEVTYCNSISVAEHVVMMILSLVR HDAEVIISQPFWPAYLTAERIAKAPKLKLALTAGIGSDHVDLQAAID--NNITVAEVTYCNSNSVAEHVVMMVLGLVR HDAEVVISQPFWPAYLTAERIAKAPKLKLALTAGIGSDHVDLQAAID--RGITVAEVTFCNSISVSEHVVMTALNLVR HDADVVISQPF PDLHVLISTPFHPAYVTAERIKKAKNLQLLLTAGIGSDHVDLKAAAA--AGLTVAEVTGSNTVSVAEDELMRILILVR EDMHVLITTPFHPAYVTAEKIKKAKTPELLLTAGIGSDHIDLPAAAA--AGLTVARVTGSNTVSVAEDELMRILILLR EDMHVLITTPFHPAYVSAERIKKAKNLELLLTAGIGSDHIDLPAAAA--AGLTVAEVTGSNTVSVAEDELMRILILLR KDAEIVITTPFFPAYISRNRIAEAPNLKLCVTAGVGSDHVDLEAANE---RKITVTEVTGSNVVSVAEHVMATILVLIR PDADIIITTPFHPAYITKERLDKAKNLKSVVVAGVGSDHIDLDYINQTGKKISVLEVTGSNVVSVAEHVVMTMLVLVR SDADVIISTPFHPAYITKERIDKAKKLKLLVVAGVGSDHIDLDYINQSGREISVLEVTGSNVVSVAEHVVMTMLVLVR EDAEIIITTPFHPGYLSAERLARAKKLKLAVTAGIGSDHVDLNAANKTNGGITVAEVTGSNVVSVAEHVLMTILVLVR EDAEIIITTPFHPGYLTAERLARAKKLKLAVTAGIGSDHVDLNAANKTNGGITVAEVTGSNVVSVAEHVLMTILVLVR A >....'..<---------- SHAYDLEAMHVGTV ARSYDVEGMHVGTV TRSYDIEGMHVGTV HRAYDLEGKTVGTV HRAYDLEGKTVGTV YRAYDLEGKTVGTV KNEYDLEDKIISTV KDAYDIEGKTIATI KDSFDIEGKVIATI KNEYDLEGKVVGTV KNEFDLEGKVVGTV 152 154 153 157 152

AAG AAG AAG GAG GAG GAG GAG GAG GAG AVG GVG GTG GVG GFG GYG GTG

C C 7 B B >. <-------------------------->..<--------> ....<-------------->............<-->..'<------------>248 RIGLAVLRRLAPFDV-HLHYTDRHRLPESVEKELN------------LTWHATREDMYPVC RIGLRVLRLLAPFDM-HLHYTDRHRLPEAVEKELN------------LTWHATREDMYGAC RIGLAVLRRFKPFGM-HLHYTDRHRLPREVELELD------------LTWHESPKDMFPAC RIGRLLLQRLKPFNC-NLLYHDRLKMDSELENQIG------------AKFEEDLDKMLSKC RYGRLLLQRLKPFNC-NLLYHDRLQINPELEKEIG------------AKFEEDLDAMLPKC RIGRLLLQRLKPFNC-NLLYHDRLKIDPELEKEIG------------AKYEEDLDAMLPKC RIGYRVLERLVAFNPKKLLYYDYQELPAEAINRLNEASKLFNGRGDIVQRVEKLEDMVAQS RIGYRVLERLLPFNPKELLYYDYQALPKEAEEKVG------------ARRVENIEELVAQA RIGYRVLERLVAFNPKELLYYDYQSLSKEAEEKVG------------ARRVHDIKELVAQA RIGERVLRRLKPFDCKELLYYDYQPLAPEVEKEIG------------CRRVDNLEEMLAQW RIGERVLRRLKPFDCKELLYYDYQPLSAEKEAEIG------------CRRVADLEEMLAQC HIGQVFMRIMEGFGA-KVIAYDIFKN 179 HIGSGLAEIFSAMGA-KVIAYDVAYN 181 SIGQALAKRAQGFDM-DIDYFDTHRA 180 HIGTQLGILAESLGM-YVYFYDIENK 184 QIGKAVIERLRGFGC-KVLAYSRSRS 179 8 ... '<------340 ISGTTLTA ISGTSLSA ISGTSLSA ISGTTIDA ISGTTIDA ISGTTIDA ISGTSLDA YSGTTLDA YSGSVIDA MSGTSLDA MSGTSLDA

D <-------- DVVTL DVVTL DVVTL DIVVI DVVVI DVIVI DVVTI DIVTV DIVTI EVVTI DVVTI

D >...<------>...<---- NCPLHPETEHMINDE NCPLHPETEHMINDE NCPLHPETEHMVNDE NTPLTEKTKGMFDKE NTPLTEKTRGMFNKE NTPLTEKTRGMFNKE NCPLHKDSRGLFNKK NAPLHAGTKGLINKE NCPLHAGSKGLVNAE NCPLHEKTRGLFNKD NCPLHEKTQGLFNKE

E E 3/10FA F F G G -------->....<---->. . <------><-------------------->'..<---->. ..... . ....<-------->.<---------------->. . TLKLFKRGAYIVNTARGKLCDRDAVARALESGRLAGYAGDVWFPQPAPKDHPWRTMPY-----NGMTPH TLKLFKRGAYLVNTARGKLCDRDAIVRALESGRLAGYAGDVWFPQPAPNDHPWRTMPH-----NGMTPH TLKLFKRGAYLVNTARGKLCDRDAVARALESGQLAGYGGDVWFPQPAPQDHPWRTMPH-----NAMTPH RIAKLKKGVLIVNNARGAIMDTQAVVDACNSGHIAGYSGDVWYPQPAPKDHPWRYMPN-----QAMTPH KIAKMKKGVIIVNNARGAIMDTQAVADACSSGHIAGYGGDVWFPQPAPKDHPWRYMPN-----HAMTPH RIAKMKKGVIIVNNARGAIMDTQAVADACSSGQVAGYGGDVWFPQPAPKDHPWRYMPN-----HAMTPH LISHMKDGAYLVNTARGAICVAEDVAEAVKSGKLAGYGGDVWDKQPAPKDHPWRTMDNKDHVGNAMTVH LLSKFKKGAWLVNTARGAICVAEDVAAALESGQLRGYGGDVWFPQPAPKDHPWRDMRNKYGAGNAMTPH LLKHFKKGAWLVNTARGAICVAEDVAAAVKSGQLRGYGGDVWFPQPAPKDHPWRSMANKYGAGNAMTPH LISKMKKGSWLVNTARGAIVVKEDVAEALKTGHLRGYGGDVWFPQPAPKDHPLRYAKNPFGGGNAMVPH LISKMKKGSWLVNTARGAIVVKEDVAEALKSGHLRGYGGDVWFPQPAPQDHPLRYAKNPFGGGNAMVPH 3/10-9A 9 10 ..<------><-->..<-- PIRDEYLIVQGGAL PIRDEYLIVQGGGL PIRDEYLIVQGGSL DFPAENYIVKDGEL EFPVENYIVKEGEL DFPVQDYIVKEGQL DYRPQDIIVQNGSY DYRPQDIILLNGEY DYRPQDIILLNGKY DYRPQDLIVHAGDY DYSPEDLIVYGGDY 9 ><------------>.................400 AGTGAHSYSKGNATGGSEEAAKFKKAV AGVGAHSYSKGNATGGSEEAAKYEKLDA AGVGAHSYSKGNATGGSEEAAKFKR AP----QYR AS----QYK AS----QYQ AT---RAYGQKK VT---KAYGKHDKK KT---KSYGADK AT---KAYGERAKITKA AT---KSYGERERAKAAAAAAKSA

8 ---------------------------------->.. QARYAAGTREILECFFEGRQTRYAAGTREILECYFEGRQARYAAGTREILECHFEGRQLRYAAGTKDMLDRYFKGEQLRYAAGVKDMLDRYFKGEQLRYAAGVKDMLDRYFKGEQKRYAQGVKNILNSYFSKKF QTRYAEGTKNILESFFTGKF QVRYAQGTKNILESFFTQKF QKRYADGTKAILESYLSGKL QKRYAAGTKAIIESYLSGKH

Figure 1. The alignment of amino acid sequences of formate dehydrogenases from bacteria (marked in green) Pseudomonas sp.101 (PseFDH, SWISS-PROT:FDH_PSESR), Moraxella sp. C1 (MorFDH, EMBL Y13245) and Paracoccus sp. 12-A (ParFDH, EMBL AB071373); mouse (light red) Mus musculus (N-terminal sequence translated from partial cDNA, EMBL AI505623); plants (cyan) ­ potato Solanum tuberosum (PotFDH, EMBL Z21493), barley Hordeum vulgare (BarFDH, EMBL D88272) and rice Oryza sativa (RicFDH, EMBL AB019533); yeasts (yellow) S.cerevisiae (SceFDH, EMBL Z75296), Candia boidinii (CboFDH, EMBL AF004096) and Pichia angusta (HanFDH, former Hansenula polymorpha, EMBL P33677); fungi (magenta) ­ Neurospora crassa (NeuFDH, EMBL L13964) and Magnaporthe grisea (MagFDH, EMBL AA415108). The amino acid sequences of some other D-specific 2-hydroxy acid dehydrogenases in the regions of coenzyme binding domains - D-lactate dehydrogenases from Lactobacillus helveticus (LheDLD, EMBL U07604) and Lactobacillus delbrueckii subsp. bulgaricus (LbuDLD, EMBL X60220), glycerate dehydrogenase from Hyphomicrobium methylovorum (HmeDGD, SWISS-PROT:DHGY_HYPME), 3-phosphoglycerate dehydrogenase from Escherishia coli (EcDPGD, EMBL L29397) and vancomycin resistance protein VanH from Enterococcus faecium (EfVanH, GeneBank M64304) are also shown. Numeration of residues and structural elements correspond to FDH from Pseudomonas sp.101. Residues of FDH active site are shown in bold red. Residues delineating substrate channel are marked in yellow background shading. Blue background shading shows "fingerprint" region and conservative Asp residue in the coenzyme binding domain. Magenta background shading shows Cys residues in PseFDH and CboFDH subjected to mutagenesis to increase chemical stability. Green background shading emphasizes active site residues in the specific protein deviating from a consensus sequence.


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D-phosphoglycerate dehydrogenase [18], D-glycerate dehydrogenase [19], D-lactate dehydrogenases [20,21] and D-hydroxy-isocaproate dehydrogenase [22] confirmed earlier assumptions and revealed strong similarities in three-dimensional fold between FDH and these proteins, Table 1 (see below section FDH internal symmetry).

3-D structure
Available structures Several high resolution structures of PseFDH are available to date: the apo-enzyme (resolution 1.80 е) [17], the ternary complex of enzyme with NAD+ and azide mimicking putative transition state (2.05 е) [17], and a complex with ADPR (1.50 е) [23]. Several other binary complexes of PseFDH (PseFDH-formate, PseFDH-NAD+, PseFDH-NADH) have been crystallized and preliminary X-ray data obtained. All the complexes had the conformation identical to the structure of apo FDH solved earlier [17]. Neither NAD+, no NADH were detected in the active centers of the solved structures suggesting that for some reasons coenzyme could not bind to the FDH active center in these crystal forms. The nature of this phenomenon is not known, one probable explanation being enzyme inactivation, e.g. oxidation of essential thiol residues that prevents entering of the coenzyme molecule into the FDH active site [3]. Structure of one of the complexes, PseFDH-formate (resolution 2.2 е), was refined to final Rf of 18.4 % [K.Polyakov, personal communication]. In the NAD+-binding pocket of FDH the formate ion was detected near the position occupied by the pyrophosphate moiety of NAD+ in the structure of holo FDH. One of the formate oxygens formed H-bond with the main chain nitrogen of Gly200. A distance between the formate carbon atom and the main chain carbonyl of Asn254 was 3.25 е, suggesting that an H-bond could be formed between formate hydrogen and this residue. No formate ions were detected neither in the putative formate binding site, no in the substrate channel (see below) suggesting that accomplished conformational change and active site rearrangement is required for productive substrate binding. The only structure of FDH from the other source found in the Protein Data Bank is the structure of a putative FDH from hyperthermophilic archaebacterium Pyrobaculum aerophilum (resolution 2.80 е) [1QP8 PDB]. This protein has been expressed in E.coli, had all the methionines substituted to selenomethionines to enable MAD technique for structure solving to be applied and carried a terminal His-tag to enable one-step purification of the protein. However there are serious doubts that the protein in question is a genuine FDH (see below). All the attempts to obtain crystals of FDH from the methylotrophic yeasts, e.g. C.boidinii, suitable for high-resolution structural analysis were up to date unsuccessful. The crystals so far obtained were of poor quality and were either twins that precluded solving of the respective structures or diffracted well above 3 е resolution range. Thus apo and holo high resolution structures of PseFDH obtained earlier remain the structural basis of our present understanding of the enzyme. FDH structure overview Details of FDH structural organisation have been extensively discussed in 1994 review [3]. Here we present the most essential features of FDH structural organization that would be required for further discussions as well as some new findings.


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+

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FDH is a typical NAD -dependent dehydrogenase composed of two identical subunits each comprising two domains: a coenzyme binding domain and a substrate binding domain based on Rossmann folds, Figure 2. The two domains are connected via two long a-helices, A and 8. The active center is situated at the domain interface and is formed by residues from only one subunit. FDH undergoes considerable conformational change on cofactor binding, as revealed by a structure of the FDH-NAD+-azide ternary complex, Figure 3. The conformational transition is accomplished via a rotation of a peripheral catalytic domains at an angle of 7.5° around hinges connecting residues 146-147 and 340-341 located in the A and 8 helices respectively. The FDH-ADPR binary complex reveals the same overall conformation as the apo FDH (r.m.s. 0.2 е) with only one minor difference in the region of a short loop Ile122Asp125, where the atoms move more than 1 е [23]. The loop advances towards the

A

B

Figure 2. Structure of PseFDH. A) FDH ternary complex with NAD+ (magenta) and formate (blue) occupying azide binding site. -helices are depicted as red cylinders (left subunit) or helices (right subunit) while -strands as cyan arrows (left) or strips (right). A long loop comprising a/a residues 12-47 present in bacterial FDHs but absent in the enzymes from other species is shown in yellow. B) Representation of the structure of the FDH subunit. Numbering of structural elements from [17].

Figure 3. Displacement of the active site residues upon the transition from apo (yellow) to holo state. Residues in the holo state are colored according to their charge: magenta ­ hydrophobic walls and His332; red ­ negatively charged; blue ­ positively charged and Ile122.


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enzyme active site in the transition state enabling Ile122 to be implicated in the substrate binding (see below). Thus revealed flexibility of the loop forms an important structural foundation for FDH catalysis. However ADPR does not induce gross structural changes comparable to those found in PseFDH-NAD+-azide ternary complex Figure 3. This suggests that the nicotinamide moiety of NAD+ is the main driving force of the conformational change giving rise to apo-holo transition and essential for transition state formation [23]. FDH internal symmetry. Conserved supersecondary structural motif in FDH and dehydrogenases of D-hydroxyacids It has long been recognized that FDH forms a highly symmetrical structure [3]. Figure 4 presents a stereo view of the two domains of FDH (coenzyme binding and catalytic) superimposed on one another. Both domains of FDH, Figure 2, have a Rossmann fold as a basic structural unit. The topology of the coenzyme binding domain of FDH - A-5-6- A-B- B-C-C-7- D-D-E- E-F- F-G-G (equivalent fragments in both domains are marked in bold underlined) is close to the classical ones found in other NAD+-dependent dehydrogenases, while the core of the catalytic domain 1-1- 4-2- 5-3- 7-4- 8-(insert of coenzyme binding domain)-8 may be regarded as a truncated copy of the coenzyme binding one. The alignment comprises vast stretches of the amino acid sequence and includes the entire -sheet of the FDH domains as well as flanking (A/8) and internal (7/2) helices. Superposition reveals 52 structurally equivalent pairs of C atoms with the r.m.s. deviation of about 1.1 е.

Figure 4. Stereo view of the superposition of the catalytic (thin line) and the coenzyme binding (thick line) domains of FDH.

When structures of the other members of the family of D-specific dehydrogenases of 2-hydroxy acids were made available it became evident (Table 1) that such a symmetrical organization is an intrinsic property of the proteins comprising the family. The catalytic domain in D-specific dehydrogenases shows a strong structural homology to the coenzyme binding domain. A topologically conserved part within D-dehydrogenase superfamily reveals a supersecondary structural motif comprising the 5-stranded lefthandedly twisted parallel -sheet with one complete and one partialRossmann fold units and two -helices, (A/8) and (7/2).


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Table 1. Superposition of the subunits of D-specific dehydrogenases (number of equivalent pairs of C atoms/r.m.s in е) (adapted from [24])
FDH FDH DGDH PGDH * DGDH 223/2.3 * DPGDH 192/2.2 232/1.4 * DLDH /~6.0 216/1.7 (186/1.3) 201/1.5 (151/0.9)

Figures in parenthesis present the alignment with the least r.m.s.

To quantitate structural similarity within the Rossmann-fold domains the parameter of the r.m.s. per aligned pair (R/N) has been suggested (Table 2) [24]. The lower the value of this parameter, i.e. the lower the r.m.s. or the higher the number of equivalent pairs - the closer is the structural relation between the proteins being compared. For very closely related proteins, e.g. apo and holo variants of the same protein the parameter is close to zero (R/N<0.005 е for the alignment of the coenzyme binding domains in apo and holo forms of FDH), while for distantly related structures (r.m.s.>2-3 е, N<20-30) the parameter R/N exceeds 0.1 е and may be considered as a cut-off above which topological similarity becomes questionable. Based on the values of R/N the Rossmann-fold domains comprising NAD+dependent dehydrogenases can be subdivided into at least three structural groups or subclasses of loosening structural similarity within the group: coenzyme binding domains of D-specific dehydrogenases - catalytic domains of D-specific dehydrogenases coenzyme binding domains of L-specific dehydrogenases, Table 2.
Table 2. Superposition of NAD(P)-binding Rossmann-fold domains (adapted from [24])
Structural Domain/ Comparison Coenzyme binding domains of D-specific dehydrogenases (CoE-D) Catalytic domains of Dspecific dehydrogenases (Cat-D) Coenzyme binding domains of L-specific dehydrogenases (CoE-L) Coenzyme binding or catalytic domains of FDH vs. reductases and flafodoxin Number of Equivalent Pairs of C Atoms, N 138±10 r.m.s., е r.m.s./N, е

0.79±0.06

0.006

70±13

1.3±0.3

0.019

53±9

1.3±0.5

0.025

28±6

1.4±0.3

0.051


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It has been also noted that essential active site residues of NAD+-dependent dehydrogenases acting on L- and D- substrates ­ arginine (Arg284 in PseFDH), histidine (His332), glutamate/aspartate (Gln313) as well as C4 position of the nicotinamide moiety of the coenzyme occupy conservative spatial positions but are interrelated by a symmetry operation reflecting difference in the configuration of a substrate molecule [25]. This observation proves that both D- and L- dehydrogenases use the same type of catalytic machinery however finely tuned to the chirality of the substrate being processed. The only FDH from the other source which structure has been reported to date, the enzyme from archaebacterium P.aerophilum, shows structural organization rather different both from PseFDH, and from the other members of D-specific dehydrogenases family. Alignment of amino acid sequences of P.aerophilum with FDHs from various sources shows only about 20 % of homology contrary to 50 % expected for distantly related FDHs. Also neither a whole molecule, no individual subunits of this protein could be superimposed over the PseFDH (r.m.s > 4 е). Moreover, the two domains comprising P.aerophilum protein did not show any structural similarity as expected among FDH/D-specific dehydrogenases subfamily. Taken altogether these facts cast a serious doubt that the P.aerophilum protein represents an FDH or even a member of the FDH family. Substrate channel The PseFDH active site is deeply buried ~15 е inside the FDH subunit and is accessible to formate anion either through the NAD+ binding site if NAD+ is absent or through a wide channel running from the active center to the surface. The amino acid residues, delineating the substrate channel are presented on Figure 1. They are highly conserved among the FDHs that emphasizes importance of the channel for proper enzyme functioning. Part of the residues comprising the channel are implicated either in substrate binding or catalysis. The channel has a barrel-like form. It is wider in the middle, up to 10-13 е, and more narrow (5-6 A) at the exits. Three side chains comprising part of the FDH active site, Arg284, His332 and to some extent Pro97 form the inner neck of the channel which makes the gateway for the substrate to proceed to the active center. The outer neck opening into the solvent is composed of Lys286, Leu257 and Tyr102. On transition to holo FDH the channel closes, the exit of the channel is additionally shielded from the solvent by the C-terminal loop Asn385-Ser390 and the inner neck separating the interior of the active center from the channel becomes narrower. Thus the interior of the active center appears to be effectively shielded from the bulk. To evaluate a mode of delivery of formate ion to the FDH active site putative substrate pathways were studied by molecular modeling using optimal biased Monte Carlo minimization algorithm (ICM software package). Regularization of the protein structure was performed (hydrogen atoms inserted and geometry of the protein molecule optimized) succeeded by mapping of the substrate- and coenzyme channels. Each channel was divided into separate sections by intersecting spheres 8 е in diameter and a search for local energy minimum for substrate-protein interactions was performed for each section of each channel by an iterative docking procedure. Position of the substrate at the enzyme active site defined as a position of azide in the structure of the holo FDH


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and verified by docking of the formate ion into the FDH active center was regarded as the end-point for both channels. Figure 5 shows energy profiles for substrate-protein interactions for each section of both channels. The benefit of energy for substrate-protein interactions is observed as the substrate advances towards the enzyme active site. For both channels the global energy minimum corresponds to the position of the formate ion in the FDH active center.

Figure 5. Substrate-protein interaction energy profiles for the coenzyme and substrate channels. The bars correspond to the consequent local energy minima of the formate anion while it advances from the bulk into the FDH active center. S marks the position corresponding to location of the formate in the enzyme active site.

In the absence of coenzyme the coenzyme channel may be used by formate to reach the enzyme active site. There are no evident steric obstacles that hinder substrate access to the active center. Simulation performed proved that there are no restrictions from molecule force field as well. Low values of local minima point to easy accessibility of this pathway. However when the coenzyme channel is occupied by NAD+ the only way for formate ion to reach the FDH active center is through the substrate channel. Figure 6 shows consecutive positions occupied by formate ion while traveling into the FDH active site. Two local energy maxima for the putative substrate channel are observed corresponding to the inner and outer necks and one profound local minimum in the middle part (less constraint part) of the channel. The main energy barrier is formed by a gate made of His332 and Arg284 ­ two of the three residues implicated in formate binding (see below). Thus flexibility of the gate and proper local conformation around this site seems to be crucial for enzyme functioning and should be accounted for when interpreting site directed mutagenesis experiments. Structural basis of enzyme specificity FDHs are strictly specific for formate. Geometry of the enzyme active center and especially of the substrate channel precludes access of any bigger molecule to the


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Vladimir O.Popov & Vladimir I.Tishkov

Figure 6. Trajectories of formate ion movement in the coenzyme and substrate channels. The position of formate with the lowest energy is shown in red. This position coincides with the location of azide anion in the FDH active site.

enzyme active site. As discussed earlier only linear and planar inorganic anions resembling either the substrate (formate) or the product (CO2) are efficient inhibitors of FDHs [3]. Even minor changes in the structure of the substrate molecule, e.g. substitution of the oxygen atom for sulfur (thioformate) results in some cases in the loss of enzymatic activity. Thioformate is a substrate for the yeast enzymes: CboFDH (Vmax ­ 18 % from the activity with formate, Km ­ 2 mM) and HpoFDH (23 % and 11 mM), while is an efficient competitive inhibitor (Ki ­ 1.5 mM) of PseFDH [26]. All studied to date FDHs have a strong preference for NAD+ as the coenzyme. The + + value of coenzyme specificity quantified as (kcat/K)NAD /(kcat/K)NADP varies from >3.109 for SceFDH (nearly absolute specificity) to 2.4.103 for PseFDH (measurable activity with high NADP+ concentrations) (see below Section V Engineering of FDH coenzyme specificity).

III. Solution studies
Main emphasis in the recent studies of FDH was put on modeling, structural characterization and genetic engineering. However some important experiments in solution have been accomplished. Two yeast FDHs from Hansenula polymorpha and Saccharomyces cerevisiae have been purified to homogeneity over the past decade and their kinetic and physicochemical properties studied in detail [27,28]. Studies in solution performed with the enzymes from these species generally confirmed the conclusions drawn earlier for other


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FDHs. Both HpoFDH and SceFDH enzymes operate via ordered Bi-Bi kinetic mechanism with NAD+ binding first [27,28]. Thus FDH from Pseudomonas sp. (PseFDH) remains the only enzyme which inhibition patterns differ from the rest of FDHs and which according to steady-state kinetic analysis operates following random addition of substrates [3]. The origin of pH-dependence of FDH kinetic parameters has been clarified using PseFDH as a sample enzyme [29]. The value of Vmax, rate-limiting hydride transfer, was nearly constant throughout the pH-range of enzyme stability (6.0 to 11.2) while the Km values for both substrates remained constant within the pH range 6 to 10. At pH values below 6 (for the coenzyme) and above 10 (for both substrate and coenzyme) the Michaelis constants increased. In the acidic range the change is attributed to the ionization of two carboxylic g