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

Area of Science:

• Microbiology and Molecular Biology
• Biochemistry and Metabolic Pathways

Background:

• Thiamine (vitamin B1) is vital for most organisms, with diverse synthesis and salvage strategies.
• The pyrimidine moiety of thiamine is typically synthesized via the ThiC pathway, but an alternative Thi5-dependent route exists.
• The Thi5-dependent pathway is poorly understood, especially in bacteria, and the yeast ortholog (ScThi5) shows limited function in heterologous systems.

Purpose of the Study:

• To investigate the role of a bacterial Thi5 ortholog in thiamine biosynthesis in Legionella pneumophila.
• To characterize the biochemical properties and functional capabilities of the bacterial HMP-P synthase (LpThi5).
• To compare the function of bacterial LpThi5 with its yeast counterpart (ScThi5) in heterologous systems.

Main Methods:

• Identified and characterized a bacterial ortholog of HMP-P synthase (Thi5) in Legionella pneumophila (LpThi5).
• Assessed the in vivo function of LpThi5 in Salmonella enterica under various growth conditions.
• Performed in vitro biochemical analyses of purified LpThi5, including substrate binding and analysis of variant proteins.

Main Results:

• A bacterial Thi5 ortholog (LpThi5) is essential for HMP-P synthesis in Legionella pneumophila.
• LpThi5 functions effectively in vivo in Salmonella enterica, contrasting with the poor performance of ScThi5.
• LpThi5 is a dimer that binds pyridoxal-5'-phosphate (PLP) and conserved residues modulate its enzymatic activity.

Conclusions:

• The Thi5-dependent pathway is a necessary route for thiamine biosynthesis in Legionella pneumophila.
• Bacterial LpThi5 represents a functional and biochemically characterized HMP-P synthase, expanding knowledge of this alternative pathway.
• Understanding LpThi5 function provides insights into metabolic network architecture and enzyme optimization in thiamine synthesis.