De Prithwiraj, Salvat Jenna, Walthers Eliza, Henriksen James, Wells Michael, Conant Richard T, Boot Claudia M
Natural Resource Ecology Laboratory, Colorado State University, Fort Collins, Colorado, United States of America.
Department of Chemistry, Colorado State University, Fort Collins, Colorado, United States of America.
PLoS One. 2025 Jun 25;20(6):e0325955. doi: 10.1371/journal.pone.0325955. eCollection 2025.
Several alditol-2-dehydrogenase enzymes from the short chain dehydrogenase (SDR) family catalyze the production of enantiopure rare ketoses from alditols as substrates and are used in biotech industry. Clearly, the absolute configuration of the internal chiral carbons in the open-chain conformation of alditols provides the structural basis for the enzymatic operation. This also allows for substrate ambiguity that manifest as enzyme promiscuity due to partial stereoselectivity of the enzyme. The issue is to make the right choice of a promiscuous enzyme and access maximum product diversity. Based on the absolute configuration of a cohort of ten enantiopure hexitols, this study is a systematic exploration of the stereochemical foundation of enzyme promiscuity in alditol-2-dehydrogenases which does not involve any kinetic analysis. Using cell-free expressed galactitol-2-dehydrogenase (G2DH), D-sorbitol-2-dehydrogenase (D-S2DH), and D-altritol-5-dehydrogenase (D-A5DH), we confirmed chemoenzymatic synthesis of all enantiopure ketohexoses through characterization by GC/MS and NMR spectroscopy. However, we found that enzyme promiscuity is beyond the partial stereoselectivity for certain alditol-2-dehydrogenase enzymes that are reliably producing enantiopure ketoses from multiple alditols. For instance, G2DH oxidizes galactitol (2R 3S; 55% conversion), a meso-alditol with a plane of symmetry, as well as L-talitol (2S 3S; 2.1% conversion) to L-tagatose. The substrate bears different absolute/relative configurations and is an example among several promiscuous oxidations. Notably, their product yields are different under similar reaction conditions indicating stereochemical preference of the enzyme. This in vitro chemoenzymatic investigation of the underlying stereochemical interplay for the enantioselective oxidation of alditols explores the potential to harness enzyme promiscuity/substrate-adaptability as a tool for the predictive synthesis of multiple enantiopure ketoses. This study focuses on unconventional stereochemical investigation into enzyme stereospecificity and promiscuity which precludes kinetic analysis. Given the role of enzyme promiscuity in evolutionary processes, systematic stereochemical analysis may prove crucial in future.
短链脱氢酶（SDR）家族中的几种醛糖醇-2-脱氢酶以醛糖醇为底物催化生成对映体纯的稀有酮糖，可用于生物技术产业。显然，醛糖醇开链构象中内部手性碳的绝对构型为酶的作用提供了结构基础。这也导致了底物的不确定性，由于酶的部分立体选择性，这种不确定性表现为酶的混杂性。问题在于如何正确选择一种混杂性酶并获得最大的产物多样性。基于一组十种对映体纯的己糖醇的绝对构型，本研究对醛糖醇-2-脱氢酶中酶混杂性的立体化学基础进行了系统探索，且不涉及任何动力学分析。使用无细胞表达的半乳糖醇-2-脱氢酶（G2DH）、D-山梨醇-2-脱氢酶（D-S2DH）和D-阿卓糖醇-5-脱氢酶（D-A5DH），我们通过气相色谱/质谱（GC/MS）和核磁共振光谱（NMR）表征，证实了所有对映体纯的酮己糖的化学酶法合成。然而，我们发现对于某些能可靠地从多种醛糖醇生成对映体纯酮糖的醛糖醇-2-脱氢酶来说，酶的混杂性超出了部分立体选择性。例如，G2DH将半乳糖醇（2R 3S；转化率55%）（一种具有对称面的内消旋醛糖醇）以及L-木糖醇（2S 3S；转化率2.1%）氧化为L-塔格糖。底物具有不同的确对映体/相对构型，是几种混杂氧化反应中的一个例子。值得注意的是，在相似反应条件下它们的产物产率不同，这表明了酶的立体化学偏好。这项关于醛糖醇对映选择性氧化潜在立体化学相互作用的体外化学酶法研究，探索了利用酶的混杂性/底物适应性作为预测合成多种对映体纯酮糖工具的潜力。本研究专注于对酶立体特异性和混杂性的非常规立体化学研究，不进行动力学分析。鉴于酶混杂性在进化过程中的作用，系统的立体化学分析在未来可能会被证明至关重要。
