Formyltetrahydrofolate hydrolase, a regulatory enzyme that functions to balance pools of tetrahydrofolate and one carbon-tetrahydrofolate adducts in Escherichia coli

Peter Lajos Nagy, Purdue University

Abstract

A gene designated purU has been identified and characterized. purU is adjacent to tyrT at min 27.7 on the Escherichia coli chromosome. The gene codes for a 280 amino acid protein. The C-terminal segment of PurU from residues 84 to 280 exhibits 27% identity with 5$\sp\prime$-phosphoribosylglycinamide (GAR) transformylase, the purN encoded enzyme catalyzing the third step of de novo purine biosynthesis. Primer extension mapping and assays of lacZ in a promoter probe vector identified two promoters giving mono- and bi-cistronic purU mRNA. Neither mRNA was regulated by purines. Based on the growth requirements of purU, purN and purU purN mutants it was proposed that PurU provides the major aerobic source of formate for an alternative, purT encoded GAR transformylase. Overexpression, purification and characterization of the enzyme established it as a 10-formyltetrahydrofolate (formyl-FH$\sb4$) hydrolase, producing FH$\sb4$ and formate in agreement with its proposed role as a formate source. Formyl-FH$\sb4$ hydrolase, a hexamer with 32-kDa subunits, is activated by methionine and inhibited by glycine. Heterotropic cooperativity is observed for activation by methionine in the presence of glycine and for inhibition by glycine in the presence of methionine. These results, along with the previous mutant analyses, lead to the conclusion that formyl-FH$\sb4$ hydrolase is a regulatory enzyme whose main function is to balance the pools of FH$\sb4$ and C1-FH$\sb4$ in response to changing growth conditions. The enzyme uses methionine and glycine to sense pools of C1-FH$\sb4$ and FH$\sb4,$ respectively. The formate released as a byproduct of its regulatory function is then used for purine biosynthesis.

Degree

Ph.D.

Advisors

Zalkin, Purdue University.

Subject Area

Microbiology|Molecular biology

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