BIOCHEMICAL PROPERTIES AND PHYLOGENY OF HYDROXYPYRUVATE REDUCTASES FROM METHANOTROPHIC BACTERIA REALIZING DIFFERENT C<sub>1</sub>-AssimiLATION PATHWAYS
In aerobic methanotrophic bacteria Methylomicrobium alcaliphilum 20Z, Methylococcus capsulatus Bath, and Methylosinus trichosporium OB3b, comparative analysis of biochemical properties of hydroxypyruvate reductase (Hpr), an indicatory enzyme of the serine pathway for assimilation of the reduced C1-compounds, was carried out. The recombinant Hpr obtained by cloning and heterologous expression of the hpr gene in Escherichia coli catalyzed NAD(P)H2-dependent reduction of hydroxypyruvate or glyoxylate, but it did not catalyze the reverse reactions of D-glycerate or glycolate oxidation. The absence of glycerate dehydrogenase activity in the methanotrophic Hpr confirmed a key role of the enzyme in utilization of C1-compounds via the serine cycle. The enzyme from Ms. trichosporium OB3b, realizing the serine cycle as the sole assimilation pathway, possessed much higher special activity and affinity in comparison to Hpr from Mm. alcaliphilum 20Z and Mc. capsulatus Bath assimilating carbon predominantly via the ribulose monophosphate (RuMP) cycle. The hpr gene was found as part of gene clusters coding the serine cycle enzymes in all sequenced methanotrophic genomes except for representatives of the Verrucomicrobia filum. Phylogenetic analyses revealed two types of hydroxypyruvate reductases: (i) Hpr of methanotrophs belonging to the Gammaproteobacteria class, which use the serine cycle along with the RMP cycle, as well as in non-methylotrophic bacteria belonging to the Alphaproteobacteria class; (ii) Hpr of methylotrophs from Alpha- and Betaproteobacteria classes that realize solely the serine cycle, and non-methylotrophic representatives of Betaproteobacteria. The putative role and origin of hydroxypyruvate reductase in methanotrophs are discussed.