Reshetnikov Alexander S, Solntseva Natalia P, Rozova Olga N, Mustakhimov Ildar I, Trotsenko Yuri A, Khmelenina Valentina N
Laboratory of Methylotrophy, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Federal Research Center "Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences", Pushchino 142290, Russia.
Department of Microbiology and Biotechnology, Pushchino State Institute of Natural Sciences, Prospect Nauki 3, Pushchino 142290, Russia.
Microorganisms. 2019 Feb 14;7(2):52. doi: 10.3390/microorganisms7020052.
The genes encoding adenosine triphosphate (ATP)- and polyphosphate (polyP)-dependent glucokinases (Glk) were identified in the aerobic obligate methanotroph sp. 12. The recombinant proteins were obtained by the heterologous expression of the genes in . ATP-Glk behaved as a multimeric protein consisting of di-, tri-, tetra-, penta- and hexamers with a subunit molecular mass of 35.5 kDa. ATP-Glk phosphorylated glucose and glucosamine using ATP (100% activity), uridine triphosphate (UTP) (85%) or guanosine triphosphate (GTP) (71%) as a phosphoryl donor and exhibited the highest activity in the presence of 5 mM Mg at pH 7.5 and 65 °C but was fully inactivated after a short-term incubation at this temperature. According to a gel filtration in the presence of polyP, the polyP-dependent Glk was a dimeric protein (2 × 28 kDa). PolyP-Glk phosphorylated glucose, mannose, 2-deoxy-D-glucose, glucosamine and -acetylglucosamine using polyP as the phosphoryl donor but not using nucleoside triphosphates. The values of ATP-Glk for glucose and ATP were about 78 μM, and the values of polyP-Glk for glucose and polyP were 450 and 21 μM, respectively. The genomic analysis of methanotrophs showed that ATP-dependent glucokinase is present in all sequenced methanotrophs, with the exception of the genera and , whereas polyP-Glks were found in all species of the genus and in only. This work presents the first characterization of polyphosphate specific glucokinase in a methanotrophic bacterium.
在需氧专性甲烷氧化菌sp. 12中鉴定出了编码三磷酸腺苷（ATP）和多聚磷酸盐（polyP）依赖性葡萄糖激酶（Glk）的基因。通过在……中对这些基因进行异源表达获得了重组蛋白。ATP-Glk表现为一种多聚体蛋白，由二聚体、三聚体、四聚体、五聚体和六聚体组成，亚基分子量为35.5 kDa。ATP-Glk以ATP（100%活性）、尿苷三磷酸（UTP）（85%）或鸟苷三磷酸（GTP）（71%）作为磷酰基供体对葡萄糖和氨基葡萄糖进行磷酸化，在pH 7.5和65℃、5 mM Mg存在的情况下表现出最高活性，但在此温度下短期孵育后会完全失活。根据在多聚磷酸盐存在下的凝胶过滤结果，多聚磷酸盐依赖性Glk是一种二聚体蛋白（2×28 kDa）。多聚磷酸盐-Glk以多聚磷酸盐作为磷酰基供体对葡萄糖、甘露糖、2-脱氧-D-葡萄糖、氨基葡萄糖和N-乙酰葡萄糖胺进行磷酸化，但不以核苷三磷酸作为磷酰基供体。ATP-Glk对葡萄糖和ATP的Km值约为78 μM，多聚磷酸盐-Glk对葡萄糖和多聚磷酸盐的Km值分别为450和21 μM。甲烷氧化菌的基因组分析表明，除了……属和……属外，所有已测序的甲烷氧化菌中均存在ATP依赖性葡萄糖激酶，而多聚磷酸盐-Glk仅在……属的所有物种以及……中被发现。这项工作首次对甲烷营养细菌中的多聚磷酸盐特异性葡萄糖激酶进行了表征。
