Bacterial and plant HAD enzymes catalyse a missing phosphatase step in thiamin diphosphate biosynthesis

Abstract

The penultimate step of thiamin diphosphate (ThDP) synthesis in plants and many bacteria is dephosphorylation of thiamin monophosphate (ThMP). Non-specific phosphatases have been thought to mediate this step and no genes encoding specific ThMP phosphatases (ThMPases) are known. Comparative genomic analysis uncovered bacterial haloacid dehalogenase (HAD) phosphatase family genes (from subfamilies IA and IB) that cluster on the chromosome with, or are fused to, thiamin synthesis genes and are thus candidates for the missing phosphatase (ThMPase). Three typical candidates (from Anaerotruncus colihominis, Dorea longicatena and Syntrophomonas wolfei) were shown to have efficient in vivo ThMPase activity by expressing them in an Escherichia coli strain engineered to require an active ThMPase for growth. In vitro assays confirmed that these candidates all preferred ThMP to any of 45 other phosphate ester substrates tested. An Arabidopsis thaliana ThMPase homologue (At4g29530) of unknown function whose expression pattern and compartmentation fit with a role in ThDP synthesis was shown to have in vivo ThMPase activity in E. coli and to prefer ThMP to any other substrate tested. However, insertional inactivation of the At4g29530 gene did not affect growth or the levels of thiamin or its phosphates, indicating that Arabidopsis has at least one other ThMPase gene. The Zea mays orthologue of At4g29530 (GRMZM2G035134) was also shown to have ThMPase activity. These data identify HAD genes specifying the elusive ThMPase activity, indicate that ThMPases are substrate-specific rather than general phosphatases and suggest that different evolutionary lineages have recruited ThMPases independently from different branches of the HAD family.

Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase

“…The same study also identified a homologous enzyme in Arabidopsis thaliana (At4g29530) that showed a similarly strong preference for ThMP. However, knocking out the At4g29530 gene did not affect growth or the levels of thiamin, ThMP, or ThDP, indicating that At4g29530 is not the major Arabidopsis ThMP phosphatase (Hasnain et al, 2016).…”

“…Our previous work raised the possibility that the At4g29530 HAD enzyme makes a minor contribution to total ThMP phosphatase activity in vivo (Hasnain et al, 2016). We therefore checked whether At4g29530 activity is a source of the residual ThMP phosphatase in th2-1 mutant plants by crossing the At4g29530 knockout (Hasnain et al, 2016) with th2-1 plants to obtain the double homozygous mutant.…”

“…Our previous work raised the possibility that the At4g29530 HAD enzyme makes a minor contribution to total ThMP phosphatase activity in vivo (Hasnain et al, 2016). We therefore checked whether At4g29530 activity is a source of the residual ThMP phosphatase in th2-1 mutant plants by crossing the At4g29530 knockout (Hasnain et al, 2016) with th2-1 plants to obtain the double homozygous mutant. Double mutant individuals did not show a more serious growth defect than th2-1 single mutants (Supplemental Figure 3), indicating that At4g29530 does not contribute significantly to the residual ThMP phosphatase activity in the th2-1 mutant.…”

“…of orthologous proteins in divergent plant species, we turned first to functional complementation of an Escherichia coli strain engineered to require an active ThMP phosphatase for growth by deleting thiL and expressing mouse thiamin diphosphokinase from a plasmid (Hasnain et al, 2016). Expression in this strain of the At5g32470 protein or either of its maize orthologs (GRMZM2G148896 and GRMZM2G078283) enabled growth on minimal medium ( Figure 4A), indicating that these proteins act as efficient ThMP phosphatases in E. coli.…”

“…However, this belief (that no dedicated ThMP phosphatase exists) has recently been overturned, for bacteria at least (Hasnain et al, 2016). Bacteria that synthesize ThDP via a plant-type pathway were found to have genes encoding HAD (haloacid dehalogenase) family phosphatases that occur in predicted operons with, or as fusions to, thiamin synthesis genes, and these HAD enzymes were found to strongly prefer ThMP to a wide range of other phosphoesters.…”