Structural insight into the catalytic mechanism of gluconate 5‐dehydrogenase from Streptococcus suis: Crystal structures of the substrate‐free and quaternary complex enzymes

Abstract

Gluconate 5-dehydrogenase (Ga5DH) is an NADP(H)-dependent enzyme that catalyzes a reversible oxidoreduction reaction between D-gluconate and 5-keto-D-gluconate, thereby regulating the flux of this important carbon and energy source in bacteria. Despite the considerable amount of physiological and biochemical knowledge of Ga5DH, there is little physical or structural information available for this enzyme. To this end, we herein report the crystal structures of Ga5DH from pathogenic Streptococcus suis serotype 2 in both substrate-free and liganded (NADP 1 /Dgluconate/metal ion) quaternary complex forms at 2.0 Å resolution. Structural analysis reveals that Ga5DH adopts a protein fold similar to that found in members of the short chain dehydrogenase/ reductase (SDR) family, while the enzyme itself represents a previously uncharacterized member of this family. In solution, Ga5DH exists as a tetramer that comprised four identical $29 kDa subunits. The catalytic site of Ga5DH shows considerable architectural similarity to that found in other enzymes of the SDR family, but the S. suis protein contains an additional residue (Arg104) that plays an important role in the binding and orientation of substrate. The quaternary complex structure provides the first clear crystallographic evidence for the role of a catalytically important serine residue and also reveals an amino acid tetrad RSYK that differs from the SYK triad found in the majority of SDR enzymes. Detailed analysis of the crystal structures reveals important contributions of Ca 21 ions to active site formation and of specific residues at the C-termini of subunits to tetramer assembly. Because Ga5DH is a potential target for therapy, our findings Additional Supporting Information may be found in the online version of this article. provide insight not only of catalytic mechanism, but also suggest a target of structure-based drug design.

Dissecting the Structural Elements for the Activation of β-Ketoacyl-(Acyl Carrier Protein) Reductase from Vibrio cholerae

“…Although it is not uncommon for a functionally competent catalytic triad (SYK) to be intact in the structures of the apo forms of some proteins annotated as 3-ketoacyl-(acyl carrier protein) reductases (PDB accession numbers 2UVD [21], 3LYL, 3OSU, 4M8S, etc. ), as well as in the structures of the apo forms of other members of the short-chain dehydrogenase superfamily (PDB accession numbers 1K2W [53], 3CXR [54], 2EWM [55], etc. ), it is unexpected that the structures of the apo forms of vcFabG and ecFabG would exhibit different conformations.…”

Metabolic Fate of Unsaturated Glucuronic/Iduronic Acids from Glycosaminoglycans

“…On the other hand, Ga5DH, which has a straight ␣-helix 4 and long ␣-helix 5, accepts the cofactor (51), although all of the above-mentioned SagDhuD residues, such as Pro 126 , Cys 149 , Gly 168 , Gly 169 , and Pro 193 , are completely conserved in Ga5DH. In this study, SagDhuD was experimentally demonstrated to exhibit an NADH-dependent reductase/dehydrogenase activity, suggesting that SagDhuD also adopts a Ga5DH-like structure through conformational changes caused by the substrate/ cofactor binding and that two structural conformations (i.e.…”

The Crystal Structure of l-Sorbose Reductase from Gluconobacter frateurii Complexed with NADPH and l-Sorbose

“…The proteins are shown as PDB accession numbers: 1X1E, TT0495 protein from Thermus thermophilus HB8; 1PR9, human L-xylulose reductase; 6 3CXT, gluconate 5-dehydrogenase from Streptococcus suis type 2. 18 carbonyl oxygen atom at C2 of L-sorbose in the ternary His116Leu-SR-NADPH-L-sorbose complex structure (details are described in the text below). On the other hand, the side chains of Tyr157 and Lys161 form hydrogen bonds with the nicotinamide ribose of NADPH.…”