98

STRUCTURAL INSIGHTS INTO THE ENZYME MECHANISM OF A
NEW FAMILY OF D-2-HYDROXYACID DEHYDROGENASES, A
CLOSE HOMOLOG OF 2-KETOPANTOATE REDUCTASE
SUKANTA MONDAL
suku@nibio.go.jp

KENJI MIZUGUCHIkenji@nibio.go.jp

National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki-shi, Osaka 5670085, JapanA newly identified family of NAD-dependent D-2-hydroxyacid dehydrogenases (D-2-HydDHs)
catalyzes the stereo-specific reduction of branched-chain 2-keto acids with bulky hydrophobic side
chains to 2-hydroxyacids. They are promising targets for industrial/practical applications,particularly in the stereo-specific synthesis of C3-branched D-hydroxyacids. Comparative modelingand docking studies have been performed to build models of the enzyme-cofactor-substratecomplexes and identify key residues for cofactor and substrate recognition. To explore large
conformational transitions (domain motions), a normal mode analysis was employed using a simple
potential and the protein models. Our analysis suggests that the new D-2-HydDH family members
possess the N-terminal NAD(H) binding Rossmann-fold domain and the α-helical C-terminal
substrate binding domain. A hinge bending motion between the N- and C-terminal domains waspredicted, which would trigger the switch of the conserved essential Lys to form a key hydrogenbond with the C2 ketone of the 2-keto acid substrates. Our findings will be useful for site-directedmutagenesis studies and protein engineering.
Keywords: Rossmann-fold; 2-keto acids; homodimer; hinge bending; normal mode analysis.

1.

Introduction

D-2-hydroxyacid dehydrogenases (D-2-HydDHs) catalyze the stereo-specific andreversible reduction of 2-ketoacids to 2-hydroxyacids. These NAD(H)-dependentoxidoreductases perform various physiological roles, with distinct preferences for thesubstrate side chains. Therefore, these enzymes are of interest in a variety of fields; they
are valuable catalysts for the production of the stereo-specific isomers of 2-hydroxyacids,
which are used in the production of semi-synthetic antibiotics or pharmaceuticals [1]; andthey are involved in the biopreservation properties of lactic acid bacteria, as certain 2hydroxyacids exhibit antifungal and antilisterial activities [13, 25].
It has been reported recently that the gene LL1323 from Lactococcus lactis encodes a
D-hydroxyacid dehydrogenase (hdhD) [4] and its homologous gene from Enterococcus
faecalis encodes a D-mandelate dehydrogenase (D-manDH2) [22]. These enzymes exhibit
high catalytic activity toward various 2-ketoacid substrates with bulky hydrophobic side
chains, particularly C3-branched substrates such as 2-ketoisocaproate (KIC), 2ketoisovalerate (KIV) and benzylformate (BEN), and strict coenzyme specificity for
NADH and NAD+. These enzymes have been shown to constitute a new family of D-2HydDHs [4, 22], which differs from the previously characterized D-2hydroxyisocaproate dehydrogenase (D-HicDH) family. The putative enzymes of this new

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family are widely distributed in various organisms and display a high diversity in theirsequences. They showed a marked sequence similarity with Escherichia coli 2ketopantoate reductase (KPR; EC 1.1.1.169), encoded by the panE gene, and previously,
these putative enzymes were incorrectly annotated as panE. KPR catalyzes the NADPHdependent reduction of ketopantoate to pantoate, an essential step in the biosynthesis of
pantothenate (vitamin B5) [5]. However, the differences in enzymatic function between
KPR and D-2-HydDH are due to only a few amino acid substitutions. Based on acomparative sequence analysis, Wada et al. [22], proposed that Asp30 and Met205 of DmanDH2 are the crucial residues for cofactor (NAD(H)) and substrate specificity.
Currently, KPR and its close homologs (non-KPRs including D-2-HydDHs) areannotated to possess the same Pfam [11] domains, namely, ApbA (PF02558) and
ApbA_C (PF08546). It has been reported that only minor structural changes, such as,amino acid replacements, cause a drastic change in the enzymatic function in D-2HydDH
family, such as, the conversions from a D-lactate dehydrogenase (D-LDH) to a D-HicDH[21], and even from a formate dehydrogenase to a D-2-HydDH [20].
Given the practical importance of these enzymes, a better understanding of thestructural basis for coenzyme and substrate specificity and the reaction mechanism would
be beneficial for protein engineering applications. In this report, we utilize comparativemodeling and bioinformatics tools and propose a novel reaction mechanism for these
enzymes.
2.

Material and Methods

2.1. Comparative Modeling and Structural Analysis
Protein sequences of the members of the family of D-2-HydDHs members were
downloaded from UniProt [6]. We have used the PDB entry 2EW2 from E. faecium(UniProt Q831Q5) as a template for modeling a representative member of the new family,
L. lactis hdhD (UniProt Q9CFY8) [4] (see section 3.1 for details). The coordinates of theanalyzed protein structures were downloaded from the RCSB Protein Data Bank (PDB)[2]. Correctly aligning the query sequence with the template is a crucial step incomparative modeling. For the present study, reliable alignments of query sequence with
template 2EW2 were generated using the sequence-structure homology recognition
software FUGUE [17]. Based on this alignment, we constructed models using
MODELLER 9v5 [10] and the quality of models was assessed with JOY [15], Verify3D[9] and Prosa-web [24]. MUSTANG [12] was used for comparing the model andtemplate structures. Dimeric interfaces were analyzed using DIMPLOT [23]. Domain
movements were analyzed with the DynDom server [14].
The structural similarity between the N- and C-terminal domains of KPR and 2EW2
enabled us to carry out molecular modeling to identify the cofactor and substrates
binding site, since the structure of the ternary complex of E. coli KPR with NADP+ andpantoate (PDB code 2OFP) has been determined [5]. The N-terminal domain of KPR

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S. Mondal & K. Mizuguchi(2OFP_A) was fitted to the modeled hdhD structure, and the NADP+ cofactor extracted
from KPR and transferred to the modeled structure. The NADP+ molecule was edited toNAD+ and NADH. Hydrogen atoms were added to the modeled complexes with the
SYBYL Biopolymer module (Tripos, 1998) and the cofactor and its neighboring aminoacid residues were optimized using the Tripos force field. Similar steps were taken formodeling substrate complexes, where the pantoate molecule was edited to KIC, KIV and
BEN. Protein-ligand interactions were studied using LIGPLOT [23] and NCONT fromthe CCP4 suit [16]. Pymol [7] was used for visualization and image generation.
2.2. Normal Mode Analysis
Normal mode analysis (NMA) of proteins is based on the hypothesis that the vibrational
normal modes exhibiting the lowest frequencies describe the largest and functionally
relevant movements in a protein. NMA has proven successful in representing domain andhinge-bending motions in proteins [18].
The PDB formatted dimeric enzyme structures were submitted to the ElNemo [19], a
web interface to the Elastic Network Model, for computing low frequency normal modes.
Input parameters included the request for the first five nontrivial normal modes, a
DQMIN to DQMAX range of -500 to 500, and a DQSTEP of 50. The NRBL setting was
set to ‘auto’, and the default cutoff of 8 Å was used to identify the elastic interaction
ranges.
3.

Results

3.1. Comparative Modeling
EF2445 from E. faecium (UniProt Q831Q5) is a member of this new family [22],although previously it had been incorrectly annotated as a putative KPR. The crystal
structure of EF2445 is available from a structural genomics project (PDB code 2EW2)
and is similar to that of E. coli KPR. E. coli KPR has been shown to adopt open andclosed conformations [5]. The structure of EF2445 is an apo (unliganded) form andshows an open conformation.
The sequence lengths of EF2445 (313 amino acids) and hdhD (312 amino acids) arenearly equal. Their full-length percentage sequence identity (PID) is 61.3. Thus, we haveused the PDB entry 2EW2 as a template and built comparative models of hdhD.
The asymmetric unit of the crystal structure of EF2445 contains two protein
molecules, burying a large amount of solvent accessible surface areas. Their dimericinterface consists of a core hydrophobic patch and a ring of polar residues. These
residues are conserved in this family (see Figure 1) and solution studies also suggest that
hdhD forms a homodimer [4].

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3.2. Hinge Bending
Although EF2445 is a homodimer, the relative orientations of the N- and C-terminal
domains are different in the two subunits. By comparing these two subunits, theDynDom server [14] detected a hinge bending motion, with the inter-domain bending
residues of 183-184. Normal mode analysis of the dimer of EF2445 also suggests acollective motions, with collectivity of 0.70 for the lowest three modes and a correlation
between predicted and experimental B-factors being 0.70 for 626 C-α atoms. Normal
mode analysis of the modeled homodimer of hdhD also reveals collective motions(Figure 2A).

Figure 1. Multiple sequence alignment of E. coli KPR (PDB code 2OFP) and representative new family
members of Enterococcus faecalis (Streptococcus faecalis) (UniProt Q831Q5, PDB code 2EW2), Clostridium
perfringens (UniProt Q0TT01), Streptococcus pyogenes (UniProt P65666), Lactococcus lactis (hdhD, UniProt
Q9CFY8), Lactobacillus sakei (UniProt Q38ZN5), Enterococcus faecium DO (UniProt Q3Y316, same as DmanDH2), Lactococcus lactis subsp. cremoris (UniProt Q02YL7), Lactobacillus casei (UniProt Q039A2),
Salmonella typhi (UniProt Q8Z4L0), Salmonella typhimurium (UniProt Q8ZN23), Staphylococcus aureus(UniProt Q2FVH3). Starting residue numbers are shown in parenthesis; GXGXXG motif, cofactor andsubstrates binding residues are shaded in gray. Dimeric interface residues of 2EW2 are marked as #. The
alignment is annotated with JOY with the following formatting convention: red, α helices; blue, strands; upper
case letters, solvent inaccessible; lower case letters, solvent accessible; bold type, hydrogen bonds to mainchain
amides; underlining, hydrogen bonds to mainchain carbonyls; italic, positive mainchain torsion angles φ.

3.3. Cofactor Recognition
hdhD and EF2445 have an Asp at position 30 on the β2-α2 loop (Figures 1 & 2B),
suggesting that they are NAD-dependent. NAD-specific enzymes commonly possess an
acidic amino acid, Asp or Glu, in the β2-α2 loop of the Rossmann fold, which forms a

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S. Mondal & K. Mizuguchihydrogen bond with the 2'-hydroxyl group of NAD, but not with the 2'-phosphate ofNADP due to electrostatic repulsion [3].
Lys72 of E. coli KPR is crucial for cofactor recognition [5], although its side chain
forms a hydrogen bond with NADP+ pyrophosphates only in the closed form. In hdhD,
the structurally equivalent Lys79 is conserved and would play a similar role (Figure 1).
Two other residues, Asn98 and Glu256 of E. coli KPR, form hydrogen bonds with the 3'hydroxyl group of nicotinamide-ribose and the equivalent residues [5], Asn105 andGlu273 of hdhD, would be crucial for cofactor recognition (Figures 1 & 2B).

Figure 2. (A) Domain movement of hdhD as seen in the first nontrivial normal mode. Proposed models forinteraction of the (B) cofactor and (C) substrates with hdhD.

3.4. Substrate Recognition
Residues Asn191, Asn195 and Ser260 of hdhD are conserved and structurally equivalent
to Asn180, Asn184 and Ser244 of E. coli KPR and these residues form hydrogen bonds
with the hydroxyacid moiety of pantoate [5]. Therefore, these residues are predicted torecognize the keto acid moiety of the 2-keto acid substrates of hdhD. Hydrophobic side
chains of hdhD substrates can be nicely accommodated in the conserved hydrophobic
pocket (Pro259, Met205, Ile254, Cys248, Met194 and Val190 of hdhD) (Figure 2C).
Consistent with this observation, a recent report suggests that a Cys residue is involved inEnzyme Mechanism of a New Family of D-2-HydDHs

103

the active center of hdhD [4]. His257 of hdhD in the C-terminal domain is structurally
equivalent to Asn241 of the E. coli KPR, which interacts with both the cofactor andsubstrate in the closed form. Although His257 in the modeled structure is >8 Å awayfrom the substrate, this residue may interact with the N-terminal domain in the closed
form and stabilize that conformation. A recent study has shown that a His reagent
significantly lowers the catalytic activity of hdhD [4] and this observation may be
explained by the predicted role of His257. However, this His residue would not be able
to interact with the keto acid moiety, even in the closed form, because of its spatial
position. No other His residue is found near the active site and therefore, no His residue
in hdhD appears to be directly involved in the catalysis, in contrast to other known D-2HydDHs, which possess the characteristic feature of His-Asp/Glu pair, as observed in DHicDH from L. casei [8].
4.

Discussion

hdhD possesses the cofactor (NAD(H)) binding N-terminal Rossmann-fold domain, andthe substrate binding α-helical C-terminal domain. Homodimerization via the C-terminal
domains appears to be important for facilitating the open-close transition, as shown in
our normal mode analysis.
Based on our comparative analysis, we have proposed a reaction mechanism for hdhD,
which is expected to hold true for the other members of this new family. The binding of
NAD(H) and the substrate induces conformational changes to provide an active
environment that favors 2-keto acids (KIC, KIV or BEN) binding. In the closed form,
Asn105 and Glu273 provide an extended hydrogen bonding network, which orients theribose-nicotinamide moiety of the cofactor in the conformation required for productive
D-substrate binding and optimal hydride transfer. In other known D-2-HydDHs, a His
residue plays an essential role in fixing the proper orientation of the substrate [8] but in
hdhD, we predict the conserved Lys187 to play an equivalent role by forming a keyhydrogen bond with the C2 ketone of 2-keto acid substrates, in analogy with KPR. The
substrate is locked during each step of the reaction via hydrogen bonds of its carboxylategroup to the conserved Ser260. Asn105 and Ly187 may play a central role in the reaction
mechanism, and Asn105, Glu273 and His257 may play a crucial role in opening andclosing the enzyme.
5.

Conclusions

NAD-dependent hdhD and other D-2-HydDH family members are of interest in a variety
of fields including the production of semi-synthetic antibiotics or pharmaceuticals andbiopreservation. Despite one unliganded (and unannotated) crystal structure, little is
known about the catalytic mechanism of the new family of hdhD. Our structure-based
analysis revealed that NAD(H) would bind in the N-terminal Rossmann-fold domain andsubstrates would bind in the α-helical C-terminal domain. A hinge bending motion
between the N- and C-terminal domains was predicted, which would trigger the switch of

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S. Mondal & K. Mizuguchi

the essential Lys187 of hdhD to form a key hydrogen bond with the substrates. We
proposed a reaction mechanism for this family based on the analogy to E. coli KPR. Ourmodels elucidated the roles of active site residues and this information would be usefulfor planning mutagenesis studies and protein engineering.
Acknowledgments
This work was in part supported by the Knowledge Cluster Initiative (stage II) by the
MEXT (Ministry of Education, Culture, Sports, Science and Technology). We thank Dr.
Lokesh P. Tripathi for carefully reading the manuscript.
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