Delineating Substrate Diversity of Disparate Short-Chain Dehydrogenase Reductase from <i>Debaryomyces hansenii</i>

<div><p>Short-chain dehydrogenase reductases (SDRs) have been utilized for catalyzing the reduction of many aromatic/aliphatic prochiral ketones to their respective alcohols. However, there is a paucity of data that elucidates their innate biological role and diverse substrate space. In this study, we executed an in-depth biochemical characterization and substrate space mapping (with 278 prochiral ketones) of an unannotated SDR (DHK) from <i>Debaryomyces hansenii</i> and compared it with structurally and functionally characterized SDR <i>Synechococcus elongatus</i>. PCC 7942 FabG to delineate its industrial significance. It was observed that DHK was significantly more efficient than FabG, reducing a diverse set of ketones albeit at higher conversion rates. Comparison of the FabG structure with a homology model of DHK and a docking of substrate to both structures revealed the presence of additional flexible loops near the substrate binding site of DHK. The comparative elasticity of the cofactor and substrate binding site of FabG and DHK was experimentally substantiated using differential scanning fluorimetry. It is postulated that the loop flexibility may account for the superior catalytic efficiency of DHK although the positioning of the catalytic triad is conserved.</p></div

Electrostatics role in substrate binding.

<p>Stereo view of electrostatic surface of DHK withmolecular docking predicted binding modes of Compound 41 (yellow sticks) and Compound 83 (green sticks). Negatively charged residues in hydrophilic sub pockets are labelled accordingly. The NADPH molecule shown as cyan sticks.</p

Purification of DHK and FabG.

<p><b>A</b>. SDS-PAGE analysis of the purified DHK. Lane 1: Purified DHK through Ni-NTA, Lane 2: Puregene Broad range marker, Lane 3: Superdex S75 purified DHK. <b>B.</b> SDS-PAGE of purified FabG Lane1: Low range marker Lane 2,3,4: gel purified FabG.</p

Co-factor binding pocket analyses.

<p>Comparison of NADPH/NADH binding pocket residues of (A) template structure yeast methylglyoxal/isovaleraldehyde reductase Gre2 (4PVD; Cyan colour), DHK (homology model; magenta), FabG from <i>Listeria monocytogenes</i> (4JRO; yellow), FabG from <i>Vibrio cholera</i> (4I08; green), and (B) FabG4 from <i>Mycobacterium tuberculosis</i> (3V1U; orange), FabG from <i>Bacillus</i> sp. (4NBU; gray). Three hotspot residues (HR1 to HR3) which are crucial in differentiating NADPH/NADH binding are highlighted as sticks and NADPH/NADH shown as yellow sticks in both the panels. Superimposition approach utilized to overlay the structures on template structures for comparison purpose.</p

Reaction Mechanism of DHK and the effect of temperature on protein stability.

<p><b>A.</b> The SDR from <i>Debaryomyces hansenii</i>, DHK,follows a compulsory random ordered reaction mechanism. In order to understand the reaction mechanism of DHK, the activity of the enzyme was monitored spectrophotometrically at 340nm by varying both the substrate and cofactor concentration under standard reaction conditions. The concentration of the substrate, Ethyl 4-chloro acetoacetate [S_B] was varied from 100μM to 1200μM and that of cofactor, NADPH [S_A] was from 50μM to 300μM. The initial velocities of the reactions were taken into account and thereby a Lineweaver-Burk graph was plotted. For a reaction mechanism to be ordered (both for compulsory as well as random) the lines in the plot was supposed to converge on the negative side. <b>B.</b> Differential scanning fluorimetry for examining the binding of cofactor and substrate with the purified enzyme. The purified enzyme has a melting temperature (Tm) of 28.04°C, when Ethyl 4-chloro acetoacetate (E4C) binds to DHK, Tm changes to 28.46°C (ΔTm = 0.42), clearly indicating that binding of E4C to the enzyme doesn’t impose stability. While when NADPH binds to DHK, Tm becomes 34.09°C (ΔTm = 6.05), giving the enzyme stability. Other cofactor, like NADH has very less affinity towards the purified protein, indicating that the enzyme prefers NADPH over NADH as a cofactor. <b>C.</b> Differential scanning fluorimetry of FabG (Tm = 58°C) with substrates NADPH (Tm = 65°C) and E4C (Tm = 59°C) (in red) and Boltzmann fit (in black).</p

Comparison of substrate space covered by DHK and FabG.

<p>The unannotated SDR from <i>Debaryomyces hansenii</i>(DHK) can reduce prochiral ketones with higher efficiency as compared to <i>Synechoccus elongatus</i> PCC7942 ß-keto acyl carrier protein reductase (FabG), having a role in fatty acid biosynthesis. (Full list in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170202#pone.0170202.s007" target="_blank">S2 Table</a>).</p

SDRs and their substrate range.

<p>Some of the reported SDRs in literature which have been tested on a limited number of substrates, thus establishing their role in the conversion of related compounds but not exploring their ability to convert a wide substrate space.</p

DHK substrate pocket analyses.

<p>Surface view of DHK sub-pockets (hydrophilic and hydrophobic) where substrate molecules interact with enzyme. Ethyl 4-chloro acetoacetate dock pose shown as sticks to highlight the catalytic site. NADPH molecule was highlighted with spheres.</p

Stereo view of substrate binding modes.

<p>Molecular docking predicted binding modes of (<b>A</b>) Ethyl 4-chloro acetoacetate, (<b>B</b>) Compound 278 (dark green sticks), Compound 73 (pale green sticks) and compound 23 (cyan sticks) with DHK protein. Active site, catalytic triad residues are highlighted with magenta and yellow colour sticks respectively. Substrate molecules in (<b>B</b>) panel were superimposed. NADPH molecule shown as cyan spheres. All the residues are labelled and hydrogen bonds specified as broken lines.</p