Rozova O N, But S Y, Khmelenina V N, Reshetnikov A S, Mustakhimov I I, Trotsenko Y A
Laboratory of Methylotrophy, Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia.
Biochemistry (Mosc). 2017 Feb;82(2):176-185. doi: 10.1134/S0006297917020092.
Two key enzymes of the ribulose monophosphate (RuMP) cycle for formaldehyde fixation, 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexulose isomerase (PHI), in the aerobic halotolerant methanotroph Methylomicrobium alcaliphilum 20Z are encoded by the genes hps and phi and the fused gene hps-phi. The recombinant enzymes HPS-His, PHI-His, and the two-domain protein HPS-PHI were obtained by heterologous expression in Escherichia coli and purified by affinity chromatography. PHI-His, HPS-His (2 × 20 kDa), and the fused protein HPS-PHI (2 × 40 kDa) catalyzed formation of fructose 6-phosphate from formaldehyde and ribulose-5-phosphate with activities of 172 and 22 U/mg, respectively. As judged from the k/K ratio, HPS-His had higher catalytic efficiency but lower affinity to formaldehyde compared to HPS-PHI. AMP and ADP were powerful inhibitors of both HPS and HPS-PHI activities. The two-domain HPS-PHI did not show isomerase activity, but the sequences corresponding to its HPS and PHI regions, when expressed separately, were found to produce active enzymes. Inactivation of the hps-phi fused gene did not affect the growth rate of the mutant strain. Analysis of annotated genomes revealed the separately located genes hps and phi in all the RuMP pathway methylotrophs, whereas the hps-phi fused gene occurred only in several methanotrophs and was absent in methylotrophs not growing under methane. The significance of these tandems in adaptation and biotechnological potential of methylotrophs is discussed.
用于固定甲醛的磷酸戊糖（RuMP）循环的两种关键酶，即6-磷酸-3-己酮糖合酶（HPS）和6-磷酸-3-己酮糖异构酶（PHI），存在于好氧耐盐甲烷氧化菌嗜碱甲基微菌20Z中，分别由基因hps和phi以及融合基因hps-phi编码。重组酶HPS-His、PHI-His和双结构域蛋白HPS-PHI通过在大肠杆菌中异源表达获得，并通过亲和层析进行纯化。PHI-His、HPS-His（2×20 kDa）和融合蛋白HPS-PHI（2×40 kDa）催化甲醛和5-磷酸核酮糖形成6-磷酸果糖，活性分别为172和22 U/mg。从k/K比值判断，与HPS-PHI相比，HPS-His具有更高的催化效率，但对甲醛的亲和力较低。AMP和ADP是HPS和HPS-PHI活性的强力抑制剂。双结构域HPS-PHI不显示异构酶活性，但当其HPS和PHI区域单独表达时，发现相应序列可产生活性酶。hps-phi融合基因的失活不影响突变菌株的生长速率。对注释基因组的分析表明，在所有RuMP途径甲基营养菌中，基因hps和phi是分开定位的，而hps-phi融合基因仅存在于几种甲烷氧化菌中，在不以甲烷为生长底物的甲基营养菌中不存在。本文讨论了这些串联基因在甲基营养菌的适应性和生物技术潜力方面的意义。
