Functional characterization of the HMP‐P synthase of Legionella pneumophila (Lpg1565)

Abstract

The production of the pyrimidine moiety in thiamine synthesis, 2‐methyl‐4‐amino‐5‐hydroxymethylpyrimidine phosphate (HMP‐P), has been described to proceed through the Thi5‐dependent pathway in Saccharomyces cerevisiae and other yeast. Previous work found that ScThi5 functioned poorly in a heterologous context. Here we report a bacterial ortholog to the yeast HMP‐P synthase (Thi5) was necessary for HMP synthesis in Legionella pneumophila. Unlike ScThi5, LpThi5 functioned in vivo in Salmonella enterica under multiple growth conditions. The protein LpThi5 is a dimer that binds pyridoxal‐5′‐phosphate (PLP), apparently without a solvent‐exposed Schiff base. A small percentage of LpThi5 protein co‐purifies with a bound molecule that can be converted to HMP. Analysis of variant proteins both in vivo and in vitro confirmed that residues in sequence motifs conserved across bacterial and eukaryotic orthologs modulate the function of LpThi5.ImportanceThiamine is an essential vitamin for the vast majority of organisms. There are multiple strategies to synthesize and salvage this vitamin. The predominant pathway for synthesis of the pyrimidine moiety of thiamine involves the Fe‐S cluster protein ThiC. An alternative pathway utilizes Thi5, a novel enzyme that uses PLP as a substrate. The Thi5‐dependent pathway is poorly characterized in yeast and has not been characterized in Bacteria. Here we demonstrate that a Thi5‐dependent pathway is necessary for thiamine biosynthesis in Legionella pneumophila and provide biochemical data to extend knowledge of the Thi5 enzyme, the corresponding biosynthetic pathway, and the role of metabolic network architecture in optimizing its function.HMP‐P synthase (Thi5) is a critical enzyme in the biosynthesis of thiamine pyrophosphate in yeast. A Thi5 homolog from Legionella pneumophila, which contributes to HMP synthesis in its native host, is an enzyme that binds pyridoxal‐5′‐phosphate and releases HMP when the purified protein is incubated with iron. In a heterologous system, Thi5 from Saccharomyces cerevisiae and L. pneumophila have functional differences that could reflect structural differences between the enzymes or metabolic differences between organismal hosts.