Characterization of hydroxymethylpyrimidine phosphate kinase from mesophilic and thermophilic bacteria and structural insights into their differential thermal stability

Area of Science:

• Biochemistry and enzymology
• Structural biology
• Microbial metabolism

Background:

• Hydroxymethylpyrimidine phosphate kinases (HMPPK), encoded by thiD, are crucial for thiamine biosynthesis, catalyzing two phosphorylations of 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP).
• Distinguishing HMPPK from homologous pyridoxal kinases (PLK), encoded by pdxK, has been challenging due to overlapping functions, particularly the first phosphorylation step.
• Limited characterization exists for mesophilic HMPPK, necessitating further study of these enzymes and their homologs.

Purpose of the Study:

• To kinetically characterize mesophilic (StHMPPK) and thermophilic (TtHMPPK) hydroxymethylpyrimidine phosphate kinases.
• To investigate the structural determinants of thermal stability in StHMPPK and TtHMPPK using molecular dynamics simulations.
• To clarify the phylogenetic relationship and distinct annotation of pyridoxal kinases (PLK) and PLK/HMPPK-like enzymes.

Main Methods:

• Kinetic characterization of Salmonella typhimurium HMPPK (StHMPPK) and Thermus thermophilus HMPPK (TtHMPPK).
• Molecular dynamics simulations to analyze structural differences and thermal stability determinants.
• Phylogenetic analysis of pyridoxal kinase and HMPPK enzyme families.

Main Results:

• Both StHMPPK and TtHMPPK exhibit specific double phosphorylation of HMP, utilizing a ternary complex mechanism and not acting on pyridoxal-like substrates.
• Phylogenetic analysis reveals PLK/HMPPK-like enzymes (from Gram-positive bacteria) are distinct from canonical PLK, despite both being encoded by pdxK.
• Molecular dynamics simulations identified significant differences in conformational flexibility between StHMPPK and TtHMPPK, correlating with hydrophobic packing and salt bridge networks, rather than hydrogen bonds, for thermal stability.

Conclusions:

• StHMPPK and TtHMPPK are confirmed HMPPK enzymes with specific double phosphorylation activity, distinct from pyridoxal kinases.
• The study clarifies the distinct evolutionary paths and necessary separate annotation of canonical PLK and PLK/HMPPK-like enzymes.
• Thermal stability in these HMPPKs is primarily governed by internal hydrophobic interactions and electrostatic networks, offering insights into enzyme adaptation to different temperatures.