Evidence that COG0325 proteins are involved in PLP homeostasis.

Pyridoxal 5'-phosphate (PLP) is an essential cofactor for nearly 60 Escherichia coli enzymes but is a highly reactive molecule that is toxic in its free form. How PLP levels are regulated and how PLP is delivered to target enzymes are still open questions. The COG0325 protein family belongs to the fold-type III class of PLP enzymes and binds PLP but has no known biochemical activity although it occurs in all kingdoms of life. Various pleiotropic phenotypes of the E. coli COG0325 (yggS) mutant have been reported, some of which were reproduced and extended in this study. Comparative genomic, genetic and metabolic analyses suggest that these phenotypes reflect an imbalance in PLP homeostasis. The E. coli yggS mutant accumulates the PLP precursor pyridoxine 5'-phosphate (PNP) and is sensitive to an excess of pyridoxine but not of pyridoxal. The pyridoxine toxicity phenotype is complemented by the expression of eukaryotic yggS orthologs. It is also suppressed by the presence of amino acids, specifically isoleucine, threonine and leucine, suggesting the PLP-dependent enzyme transaminase B (IlvE) is affected. These genetic results lay a foundation for future biochemical studies of the role of COG0325 proteins in PLP homeostasis.

Plant-driven repurposing of the ancient S-adenosylmethionine repair enzyme homocysteine S-methyltransferase.

Homocysteine S-methyltransferases (HMTs) are widely distributed enzymes that convert homocysteine (Hcy) into methionine (Met) using either S-adenosylmethionine (AdoMet) or the plant secondary product S-methylmethionine (SMM) as methyl donor. AdoMet is chirally and covalently unstable, with racemization of natural (S,S)-AdoMet yielding biologically inactive (R,S)-AdoMet and depurination yielding S-ribosylmethionine (S-ribosylMet). The apparently futile AdoMet-dependent reaction of HMTs was assigned a role in repairing chiral damage to AdoMet in yeast: yeast HMTs strongly prefer (R,S)- to (S,S)-AdoMet and thereby limit (R,S)-AdoMet build-up [Vinci and Clarke (2010) J. Biol. Chem. 285, 20526-20531]. In the present study, we show that bacterial, plant, protistan and animal HMTs likewise prefer (R,S)- over (S,S)-AdoMet, that their ability to use SMM varies greatly and is associated with the likely prevalence of SMM in the environment of the organism and that most HMTs cannot use S-ribosylMet. Taken with results from comparative genomic and phylogenetic analyses, these data imply that (i) the ancestral function of HMTs was (R,S)-AdoMet repair, (ii) the efficient use of SMM reflects the repurposing of HMTs after the evolutionary advent of plants introduced SMM into the biosphere, (iii) this plant-driven repurposing was facile and occurred independently in various lineages, and (iv) HMTs have little importance in S-ribosylMet metabolism.