Laboratory for Microbial Biochemistry, Philipps University of Marburg, 35043, Marburg, Germany.
Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, 30-239, Krakow, Poland.
Appl Microbiol Biotechnol. 2024 Jul 8;108(1):410. doi: 10.1007/s00253-024-13225-z.
We characterise a reversible bacterial zinc-containing benzyl alcohol dehydrogenase (BaDH) accepting either NAD or NADP as a redox cofactor. Remarkably, its redox cofactor specificity is pH-dependent with the phosphorylated cofactors favored at lower and the dephospho-forms at higher pH. BaDH also shows different steady-state kinetic behavior with the two cofactor forms. From a structural model, the pH-dependent shift may affect the charge of a histidine in the 2'-phosphate-binding pocket of the redox cofactor binding site. The enzyme is phylogenetically affiliated to a new subbranch of the Zn-containing alcohol dehydrogenases, which share this conserved residue. BaDH appears to have some specificity for its substrate, but also turns over many substituted benzyl alcohol and benzaldehyde variants, as well as compounds containing a conjugated C=C double bond with the aldehyde carbonyl group. However, compounds with an sp-hybridised C next to the alcohol/aldehyde group are not or only weakly turned over. The enzyme appears to contain a Zn in its catalytic site and a mixture of Zn and Fe in its structural metal-binding site. Moreover, we demonstrate the use of BaDH in an enzyme cascade reaction with an acid-reducing tungsten enzyme to reduce benzoate to benzyl alcohol. KEY POINTS: •Zn-containing BaDH has activity with either NAD or NADP at different pH optima. •BaDH converts a broad range of substrates. •BaDH is used in a cascade reaction for the reduction of benzoate to benzyl alcohol.
我们描述了一种可逆的含锌细菌苄醇脱氢酶（BaDH），它可以接受 NAD 或 NADP 作为氧化还原辅助因子。值得注意的是，其氧化还原辅助因子特异性依赖于 pH，在较低 pH 下更倾向于磷酸化形式，而在较高 pH 下则更倾向于去磷酸化形式。BaDH 还表现出与两种辅助因子形式不同的稳态动力学行为。根据结构模型，pH 依赖性变化可能会影响氧化还原辅助因子结合位点 2'-磷酸结合口袋中组氨酸的电荷。该酶在系统发育上与含锌醇脱氢酶的一个新分支相关，该分支共享这个保守残基。BaDH 似乎对其底物具有一定的特异性，但也能转化许多取代的苄醇和苯甲醛变体，以及含有与醛羰基共轭 C=C 双键的化合物。然而，与醇/醛基相邻的 sp 杂化 C 上的化合物则不能或仅微弱转化。该酶似乎在其催化位点中含有一个 Zn，在其结构金属结合位点中含有 Zn 和 Fe 的混合物。此外，我们证明了 BaDH 在与酸还原钨酶的酶级联反应中的应用，以将苯甲酸还原为苄醇。关键点： •含 Zn 的 BaDH 在不同 pH 最佳条件下具有 NAD 或 NADP 的活性。 •BaDH 转化广泛的底物。 •BaDH 用于级联反应将苯甲酸还原为苄醇。
