Segawa Mutsumi, Wen Cheng, Orita Izumi, Nakamura Satoshi, Fukui Toshiaki
School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
J Biosci Bioeng. 2019 Mar;127(3):294-300. doi: 10.1016/j.jbiosc.2018.08.009. Epub 2018 Sep 19.
Ralstonia eutropha H16 contains both NADH- and NADPH-dependent reduction activities to acetoacetyl-CoA, and the NADPH-dependent activity is mediated by PhaB paralogs with (R)-stereospecificity providing (R)-3-hydroxybutyryl (3HB)-CoA monomer for poly((R)-3-hydroxybutyrate) synthesis. In contrast, the gene encoding the NADH-dependent enzyme has not been identified to date. This study focused on the NADH-dependent dehydrogenase with (S)-stereospecificity in R. eutropha, as the (S)-specific reduction of acetoacetyl-CoA potentially competed with the polyester biosynthesis via (R)-3HB-CoA. The NADH-dependent reduction activity decreased to one-half when the gene for H16_A0282 (PaaH1), one of two homologs of clostridial NADH-3HB-CoA dehydrogenase, was deleted. The enzyme responsible for the remaining activity was partially purified and identified as H16_A0602 (Had) belonging to a different family from PaaH1. Gene disruption analysis elucidated that most of the NADH-dependent activity was mediated by PaaH1 and Had. The kinetic analysis using the recombinant enzymes indicated that PaaH1 and Had were both NADH-dependent 3-hydroxyacyl-CoA dehydrogenases with rather broad substrate specificity to 3-oxoacyl-CoAs of C to C. The deletion of had in the R. eutropha strain previously engineered for biosynthesis of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) led to decrease in the C composition of the copolyester synthesized from soybean oil, suggesting the role of Had in (S)-specific reduction of 3-oxohexanoyl-CoA with reverse β-oxidation direction. Crotonase ((S)-specific enoyl-CoA hydratase) in R. eutropha H16 was also partially purified and identified as H16_A3307.
真养产碱杆菌H16对乙酰乙酰辅酶A同时具有依赖NADH和NADPH的还原活性，且依赖NADPH的活性由具有（R）-立体特异性的PhaB旁系同源物介导，为聚（（R）-3-羟基丁酸酯）合成提供（R）-3-羟基丁酰（3HB）-辅酶A单体。相比之下，编码依赖NADH的酶的基因迄今尚未被鉴定出来。本研究聚焦于真养产碱杆菌中具有（S）-立体特异性的依赖NADH的脱氢酶，因为乙酰乙酰辅酶A的（S）-特异性还原可能会与通过（R）-3HB-辅酶A的聚酯生物合成产生竞争。当梭菌NADH-3HB-辅酶A脱氢酶的两个同源物之一H16_A0282（PaaH1）的基因被缺失时，依赖NADH的还原活性降低到了一半。负责剩余活性的酶被部分纯化，并鉴定为H16_A0602（Had），它属于与PaaH1不同的家族。基因破坏分析表明，大部分依赖NADH的活性由PaaH1和Had介导。使用重组酶进行的动力学分析表明，PaaH1和Had都是依赖NADH的3-羟基酰基辅酶A脱氢酶，对C至C的3-氧代酰基辅酶A具有相当广泛的底物特异性。在先前为聚（（R）-3-羟基丁酸酯-co-（R）-3-羟基己酸酯）生物合成而改造的真养产碱杆菌菌株中缺失had，导致由大豆油合成的共聚酯的C组成减少，这表明Had在以反向β-氧化方向对3-氧代己酰辅酶A进行（S）-特异性还原中发挥作用。真养产碱杆菌H16中的巴豆酸酶（（S）-特异性烯酰辅酶A水合酶）也被部分纯化，并鉴定为H16_A3307。
