Structural, Biochemical, and Evolutionary Characterizations of Glyoxylate/Hydroxypyruvate Reductases Show Their Division into Two Distinct Subfamilies.

The d-2-hydroxyacid dehydrogenase (2HADH) family illustrates a complex evolutionary history with multiple lateral gene transfers and gene duplications and losses. As a result, the exact functional annotation of individual members can be extrapolated to a very limited extent. Here, we revise the previous simplified view on the classification of the 2HADH family; specifically, we show that the previously delineated glyoxylate/hydroxypyruvate reductase (GHPR) subfamily consists of two evolutionary separated GHRA and GHRB subfamilies. We compare two representatives of these subfamilies from Sinorhizobium meliloti (SmGhrA and SmGhrB), employing a combination of biochemical, structural, and bioinformatics approaches. Our kinetic results show that both enzymes reduce several 2-ketocarboxylic acids with overlapping, but not equivalent, substrate preferences. SmGhrA and SmGhrB show highest activity with glyoxylate and hydroxypyruvate, respectively; in addition, only SmGhrB reduces 2-keto-d-gluconate, and only SmGhrA reduces pyruvate (with low efficiency). We present nine crystal structures of both enzymes in apo forms and in complexes with cofactors and substrates/substrate analogues. In particular, we determined a crystal structure of SmGhrB with 2-keto-d-gluconate, which is the biggest substrate cocrystallized with a 2HADH member. The structures reveal significant differences between SmGhrA and SmGhrB, both in the overall structure and within the substrate-binding pocket, offering insight into the molecular basis for the observed substrate preferences and subfamily differences. In addition, we provide an overview of all GHRA and GHRB structures complexed with a ligand in the active site.

Biochemical Properties and Phylogeny of Hydroxypyruvate Reductases from Methanotrophic Bacteria with Different C1-Assimilation Pathways.

In the aerobic methanotrophic bacteria Methylomicrobium alcaliphilum 20Z, Methylococcus capsulatus Bath, and Methylosinus trichosporium OB3b, the biochemical properties of hydroxypyruvate reductase (Hpr), an indicator enzyme of the serine pathway for assimilation of reduced C1-compounds, were comparatively analyzed. The recombinant Hpr obtained by cloning and heterologous expression of the hpr gene in Escherichia coli catalyzed NAD(P)H-dependent reduction of hydroxypyruvate or glyoxylate, but did not catalyze the reverse reactions of D-glycerate or glycolate oxidation. The absence of the 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 a sole assimilation pathway had 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 the representatives of the Verrucomicrobia phylum. Phylogenetic analyses revealed two types of Hpr: (i) Hpr of methanotrophs belonging to the Gammaproteobacteria class, which use the serine cycle along with the RuMP cycle, as well as of non-methylotrophic bacteria belonging to the Alphaproteobacteria class; (ii) Hpr of methylotrophs from Alpha- and Betaproteobacteria classes that use only the serine cycle and of non-methylotrophic representatives of Betaproteobacteria. The putative role and origin of hydroxypyruvate reductase in methanotrophs are discussed.