Rozova Olga N, Khmelenina Valentina N, Gavletdinova Juliya Z, Mustakhimov Ildar I, Trotsenko Yuri A
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, Pushchino, 142290, Russia,
Antonie Van Leeuwenhoek. 2015 Oct;108(4):965-74. doi: 10.1007/s10482-015-0549-5. Epub 2015 Aug 15.
Recombinant acetate kinase (AcK) was obtained from the aerobic haloalkalitolerant methanotroph Methylomicrobium alcaliphilum 20Z by heterologous expression in Escherichia coli and purification by affinity chromatography. The substrate specificity, the kinetics and oligomeric state of the His6-tagged AcK were determined. The M. alcaliphilum AcK (2 × 45 kDa) catalyzed the reversible phosphorylation of acetate into acetyl phosphate and exhibited a dependence on Mg(2+) or Mn(2+) ions and strong specificity to ATP/ADP. The enzyme showed the maximal activity and high stability at 70 °C. AcK was 20-fold more active in the reaction of acetate synthesis compared to acetate phosphorylation and had a higher affinity to acetyl phosphate (K m 0.11 mM) than to acetate (K m 5.6 mM). The k cat /K m ratios indicated that the enzyme had a remarkably high catalytic efficiency for acetate and ATP formation (k cat/K m = 1.7 × 10(6)) compared to acetate phosphorylation (k cat/K m = 2.5 × 10(3)). The ack gene of M. alcaliphilum 20Z was shown to be co-transcribed with the xfp gene encoding putative phosphoketolase. The Blast analysis revealed the ack and xfp genes in most genomes of the sequenced aerobic methanotrophs, as well as methylotrophic bacteria not growing on methane. The distribution and metabolic role of the postulated phosphoketolase shunted glycolytic pathway in aerobic C1-utilizing bacteria is discussed.
通过在大肠杆菌中进行异源表达并经亲和层析纯化，从好氧嗜盐碱甲烷氧化菌嗜碱甲基微菌20Z中获得了重组乙酸激酶（AcK）。测定了His6标签化的AcK的底物特异性、动力学和寡聚状态。嗜碱甲基微菌AcK（2×45 kDa）催化乙酸可逆磷酸化生成乙酰磷酸，表现出对Mg(2+)或Mn(2+)离子的依赖性以及对ATP/ADP的强特异性。该酶在70°C时表现出最大活性和高稳定性。与乙酸磷酸化相比，AcK在乙酸合成反应中的活性高20倍，并且对乙酰磷酸（K m 0.11 mM）的亲和力高于对乙酸（K m 5.6 mM）的亲和力。k cat /K m比值表明，与乙酸磷酸化（k cat/K m = 2.5×10(3)）相比，该酶对乙酸和ATP形成具有极高的催化效率（k cat/K m = 1.7×10(6)）。嗜碱甲基微菌20Z的ack基因被证明与编码假定磷酸酮醇酶的xfp基因共转录。Blast分析揭示了大多数已测序好氧甲烷氧化菌以及不以甲烷为食的甲基营养细菌的基因组中都存在ack和xfp基因。本文讨论了假定的磷酸酮醇酶分流糖酵解途径在好氧利用C1细菌中的分布和代谢作用。
