Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629HZ, Delft, the Netherlands.
J Biotechnol. 2019 Dec 20;306:142-148. doi: 10.1016/j.jbiotec.2019.10.005. Epub 2019 Oct 4.
The tungsten containing Aldehyde:ferredoxin oxidoreductases (AOR) offer interesting opportunities for biocatalytic approaches towards aldehyde oxidation and carboxylic acid reduction. The hyperthermophilic archaeon Pyrococcus furiosus encodes five different AOR family members: glyceraldehyde-3-phosphate oxidoreductase (GAPOR), aldehyde oxidoreductase (AOR), and formaldehyde oxidoreductase (FOR), WOR4 and WOR5. GAPOR functions as a glycolytic enzyme and is highly specific for the substrate glyceraldehyde-3-phosphate (GAP). AOR, FOR and WOR5 have a broad substrate spectrum, and for WOR4 no substrate has been identified to date. As ambiguous kinetic parameters have been reported for different AOR family enzymes the steady state kinetics under different physiologically relevant conditions was explored. The GAPOR substrate GAP was found to degrade at 60 °C by non-enzymatic elimination of the phosphate group to methylglyoxal with a half-life t = 6.5 min. Methylglyoxal is not a substrate or inhibitor of GAPOR. D-GAP was identified as the only substrate oxidized by GAPOR, and the kinetics of the enzyme was unaffected by the presence of L-GAP, which makes GAPOR the first enantioselective enzyme of the AOR family. The steady-state kinetics of GAPOR showed partial substrate inhibition, which assumes the GAP inhibited form of the enzyme retains some activity. This inhibition was found to be alleviated completely by a 1 M NaCl resulting in increased enzyme activity at high substrate concentrations. GAPOR activity was strongly pH dependent, with the optimum at pH 9. At pH 9, the substrate is a divalent anion and, therefore, positively charged amino acid residues are likely to be involved in the binding of the substrate. FOR exhibited a significant primary kinetic isotope effect of the apparent Vmax for the deuterated substrate, formaldehyde-d, which shows that the rate-determining step involves a CH bond break from the aldehyde. The implications of these results for the reaction mechanism of tungsten-containing AORs, are discussed.
铁氧还蛋白氧化还原酶（AOR）为醛氧化和羧酸还原的生物催化方法提供了有趣的机会。 高温古菌 Pyrococcus furiosus 编码五个不同的 AOR 家族成员：甘油醛-3-磷酸氧化还原酶（GAPOR）、醛氧化还原酶（AOR）和甲醛氧化还原酶（FOR）、WOR4 和 WOR5。 GAPOR 作为糖酵解酶起作用，并且对底物甘油醛-3-磷酸（GAP）具有高度特异性。 AOR、FOR 和 WOR5 具有广泛的底物谱，而 WOR4 迄今为止尚未鉴定出任何底物。由于不同的 AOR 家族酶报道了不明确的动力学参数，因此探索了不同生理相关条件下的稳态动力学。发现 GAPOR 的底物 GAP 在 60°C 下通过磷酸基团非酶促消除为甲基乙二醛而降解，半衰期 t = 6.5 分钟。甲基乙二醛不是 GAPOR 的底物或抑制剂。D-GAP 被鉴定为唯一被 GAPOR 氧化的底物，并且酶的动力学不受 L-GAP 的存在影响，这使得 GAPOR 成为 AOR 家族的第一个对映选择性酶。GAPOR 的稳态动力学表现出部分底物抑制，这表明 GAP 抑制形式的酶保留一些活性。通过 1M NaCl 完全缓解了这种抑制，从而在高底物浓度下增加了酶的活性。GAPOR 活性强烈依赖于 pH 值，最适 pH 值为 9。在 pH 9 时，底物是二价阴离子，因此，带正电荷的氨基酸残基可能参与了底物的结合。FOR 对氘代底物甲醛-d 的表观 Vmax 表现出显著的一级动力学同位素效应，这表明速率决定步骤涉及醛的 CH 键断裂。讨论了这些结果对含钨 AOR 反应机制的影响。
