Losey Nathaniel A, Mus Florence, Peters John W, Le Huynh M, McInerney Michael J
Department of Plant Biology and Microbiology, University of Oklahoma, Norman, Oklahoma, USA.
Department of Chemistry and Biochemistry, Institute of Biological Chemistry, Washington State University, Pullman, Washington, USA.
Appl Environ Microbiol. 2017 Sep 29;83(20). doi: 10.1128/AEM.01335-17. Print 2017 Oct 15.
syntrophically oxidizes short-chain fatty acids (four to eight carbons in length) when grown in coculture with a hydrogen- and/or formate-using methanogen. The oxidation of 3-hydroxybutyryl-coenzyme A (CoA), formed during butyrate metabolism, results in the production of NADH. The enzyme systems involved in NADH reoxidation in are not well understood. The genome of contains a multimeric [FeFe]-hydrogenase that may be a mechanism for NADH reoxidation. The genes for the multimeric [FeFe]-hydrogenase (; SWOL_RS05165, SWOL_RS05170, SWOL_RS05175) and [FeFe]-hydrogenase maturation proteins (SWOL_RS05180, SWOL_RS05190, SWOL_RS01625) were coexpressed in , and the recombinant Hyd1ABC was purified and characterized. The purified recombinant Hyd1ABC was a heterotrimer with an αβγ configuration and a molecular mass of 115 kDa. Hyd1ABC contained 29.2 ± 1.49 mol of Fe and 0.7 mol of flavin mononucleotide (FMN) per mole enzyme. The purified, recombinant Hyd1ABC reduced NAD and oxidized NADH without the presence of ferredoxin. The HydB subunit of the multimeric [FeFe]-hydrogenase lacks two iron-sulfur centers that are present in known confurcating NADH- and ferredoxin-dependent [FeFe]-hydrogenases. Hyd1ABC is a NADH-dependent hydrogenase that produces hydrogen from NADH without the need of reduced ferredoxin, which differs from confurcating [FeFe]-hydrogenases. Hyd1ABC provides a mechanism by which can reoxidize NADH produced during syntrophic butyrate oxidation when low hydrogen partial pressures are maintained by a hydrogen-consuming microorganism. Our work provides mechanistic understanding of the obligate metabolic coupling that occurs between hydrogen-producing fatty and aromatic acid-degrading microorganisms and their hydrogen-consuming partners in the process called syntrophy (feeding together). The multimeric [FeFe]-hydrogenase used NADH without the involvement of reduced ferredoxin. The multimeric [FeFe]-hydrogenase would produce hydrogen from NADH only when hydrogen concentrations were low. Hydrogen production from NADH by would likely cease before any detectable amount of cell growth occurred. Thus, continual hydrogen production requires the presence of a hydrogen-consuming partner to keep hydrogen concentrations low and explains, in part, the obligate requirement that has for a hydrogen-consuming partner organism during growth on butyrate. We have successfully expressed genes encoding a multimeric [FeFe]-hydrogenase in , demonstrating that such an approach can be advantageous to characterize complex redox proteins from difficult-to-culture microorganisms.
与利用氢气和/或甲酸的产甲烷菌共培养时，[具体微生物名称]能以互营方式氧化短链脂肪酸（长度为4至8个碳）。丁酸代谢过程中形成的3-羟基丁酰辅酶A（CoA）的氧化会产生NADH。参与[具体微生物名称]中NADH再氧化的酶系统尚不清楚。[具体微生物名称]的基因组包含一种多聚体[FeFe]-氢化酶，这可能是NADH再氧化的一种机制。多聚体[FeFe]-氢化酶（[具体微生物名称]；SWOL_RS05165、SWOL_RS05170、SWOL_RS05175）和[FeFe]-氢化酶成熟蛋白（SWOL_RS05180、SWOL_RS05190、SWOL_RS01625）的基因在[具体宿主名称]中共表达，纯化并表征了重组Hyd1ABC。纯化后的重组Hyd1ABC是一种具有αβγ构型、分子量为115 kDa的异源三聚体。每摩尔酶，Hyd1ABC含有29.2±1.49摩尔铁和0.7摩尔黄素单核苷酸（FMN）。纯化后的重组Hyd1ABC在没有铁氧化还原蛋白的情况下能还原NAD并氧化NADH。[具体微生物名称]的多聚体[FeFe]-氢化酶的HydB亚基缺少已知的依赖NADH和铁氧化还原蛋白的[FeFe]-氢化酶中存在的两个铁硫中心。Hyd1ABC是一种依赖NADH的氢化酶，可在不需要还原型铁氧化还原蛋白的情况下从NADH产生氢气，这与已知的[FeFe]-氢化酶不同。Hyd1ABC提供了一种机制，当氢气消耗微生物维持低氢气分压时，[具体微生物名称]可以再氧化互营丁酸氧化过程中产生的NADH。我们的工作提供了对产氢脂肪酸和芳香酸降解微生物与其氢气消耗伙伴在称为互营共生（共同进食）过程中发生的专性代谢偶联的机制理解。多聚体[FeFe]-氢化酶在没有还原型铁氧化还原蛋白参与的情况下利用NADH。多聚体[FeFe]-氢化酶仅在氢气浓度低时才会从NADH产生氢气。[具体微生物名称]从NADH产生氢气的过程可能在任何可检测到的细胞生长发生之前就停止了。因此，持续产生氢气需要存在氢气消耗伙伴以保持氢气浓度低，这部分解释了[具体微生物名称]在以丁酸为生长底物时对氢气消耗伙伴生物体的专性需求。我们已成功在[具体宿主名称]中表达了编码多聚体[FeFe]-氢化酶的基因，表明这种方法对于表征来自难以培养的微生物的复杂氧化还原蛋白可能是有利的。
