A 5-formyltetrahydrofolate cycloligase paralog from all domains of life: comparative genomic and experimental evidence for a cryptic role in thiamin metabolism

Abstract

A paralog (here termed COG0212) of the ATP-dependent folate salvage enzyme 5-formyltetrahydrofolate cycloligase (5-FCL) occurs in all domains of life and, although typically annotated as 5-FCL in pro- and eukaryotic genomes, is of unknown function. COG0212 is similar in overall structure to 5-FCL, particularly in the substrate binding region, and has distant similarity to other kinases. The Arabidopsis thaliana COG0212 protein was shown to be targeted to chloroplasts and to be required for embryo viability. Comparative genomic analysis revealed that a high proportion (19%) of archaeal and bacterial COG0212 genes are clustered on the chromosome with various genes implicated in thiamin metabolism or transport but showed no such association between COG0212 and folate metabolism. Consistent with the bioinformatic evidence for a role in thiamin metabolism, ablating COG0212 in the archaeon Haloferax volcanii caused accumulation of thiamin monophosphate. Biochemical and functional complementation tests of several known and hypothetical thiamin-related activities (involving thiamin, its breakdown products, and their phosphates) were, however, negative. Also consistent with the bioinformatic evidence, the COG0212 proteins from A. thaliana and prokaryote sources lacked 5-FCL activity in vitro and did not complement the growth defect or the characteristic 5-formyltetrahydrofolate accumulation of a 5-FCL-deficient (ΔygfA) Escherichia coli strain. We therefore propose (a) that COG0212 has an unrecognized yet sometimes crucial role in thiamin metabolism, most probably in salvage or detoxification, and (b) that is not a 5-FCL and should no longer be so annotated.

A cross-kingdom Nudix enzyme that pre-empts damage in thiamin metabolism

“…First, the thiamin moiety of ThDP is chemically and enzymatically labile [15,25], so that ThDP can give rise directly to damaged diphosphates such as oxoThDP and oxyThDP. Such damaged diphosphates have the same potential repercussions as diphosphates made by TDPK.…”

“…It is important to note that there are potentially many others, for oxy-and oxo-thiamin and their diphosphates are convenient model compounds from a large, but poorly known, range of damaged forms of thiamin. Other damaged forms include desthiothiamin [41] and mixed thiamin disulphides [15], all of which could exist as diphosphates. It is not known which damaged forms of ThDP predominate in vivo; whichever these are, they may be better substrates for Tnr3 and its orthologues than oxy-or oxo-ThDP.…”

“…oxoThDP (oxothiamin diphosphate) and oxyThDP were synthesized using mouse TDPK (mTDPK) as described previously [15] with the following modifications. Reactions (100 μl) contained 50 mM Tris/HCl (pH 8.0), 15 mM MgCl 2 , 10 mM ATP, 10 mM oxothiamin or oxythiamin, and 52-130 μg of mTDPK.…”

“…OxyThDP and oxoThDP yields were 82 % and 100 % respectively. The products of the reaction were directly used for enzymatic assays after removal of TDPK protein as described previously [15]. As the oxyThDP yield was only 82 % the amount of deproteinized reaction mixture added to assays was adjusted accordingly.…”

“…Since Nudix enzymes can have higher ('housecleaning') activity with damaged substrates than with native ones [9], we tested the diphosphates of oxothiamin and oxythiamin. Oxothiamin is an oxidation product of thiamin [15] and oxythiamin is a hydrolysis product [25] (Figure 1). Both oxoThDP and oxyThDP were hydrolysed, yielding orthophosphate, far more rapidly (6-60-fold) than ThDP by Tnr3, Yjr142w and AtNUDT20, and were also hydrolysed by ZmNUDIX.…”