FUNCTIONAL AND STRUCTURAL BASIS FOR THE EMERGENCE OF PYRIDOXAL KINASE IN THE BACTERIAL THID VITAMIN KINASE SUBFAMILY

Thiamine biosynthesis requires phosphorylation of 4-amino-2-hydroxymethyl-5-methylpyrimidine (HMP) to HMP-PP via the intermediate HMP-P. In bacteria, two homologous enzymes catalyze HMP phosphorylation: (1) ThiD-HMPPK kinases, which phosphorylate both HMP and HMP-P, and (2) ThiD2-ccPLK/HMPK enzymes, which lack HMP-P kinase activity but also phosphorylate pyridoxal (PL). Crystal structures of an ancestral ThiD-HMPPK from Enterobacterales (ancEn) reveal residues H179 and T211 as critical for HMP-P phosphorylation residues that are absent in ThiD2-ccPLK/HMPK. Conversely, the crystal structure of Staphylococcus aureus ThiD2-ccPLK/HMPK shows a hemithioacetal intermediate formed between PL and the conserved residue C111.

Phylogenetic analyses suggest that ThiD2-ccPLK/HMPK enzymes evolved from ThiD-HMPPKs, accompanied by a loss of HMP-P kinase activity and the emergence of PL kinase activity. To investigate this functional shift, we reconstructed three ancestral enzymes: ancThiD (ThiD-HMPPK ancestor), ancC (intermediate), and ancThiD2 (ThiD2-ccPLK/HMPK ancestor). In silico analyses support a stepwise evolutionary trajectory beginning with T211A, followed by Q44M, A111C, and H179S substitutions, reshaping the active site.

Biochemical characterization revealed that ancThiD phosphorylates only HMP and HMP-P, with low PL binding affinity. ancC exhibits broadened substrate specificity, efficiently phosphorylating HMP, HMP-P, and gain the capability to phosphorylate PL. In ancThiD2, PL kinase activity is fully optimized, becoming the most efficient activity, while HMP-P phosphorylation remained residual.

Finally, structural and molecular dynamics analyses revealed increased active-site loop flexibility in the presence of PL for ancThiD and ancC but decreased flexibility in ancThiD2. Functionally, ancThiD2C111A mutant reduced catalytic efficiency for PL by eightfold, while the ancCA110C mutation had no effect on the catalytic efficiency for PL due to compensatory changes in K_M and k_cat for PL, suggesting that other substitutions contributed to the fully optimization of PL phosphorylation in ccThiD2 enzymes.

This work was supported by ANID Fellowship for PhD complementary funds 21221449 (Nicolás Fuentes-Ugarte), FONDECYT 1221667 (Víctor Castro-Fernandez).