Biochemical Investigation of PumG, a Key Pyridoxal 5′-Phosphate (PLP)-Dependent Aminotransferase Involved in Pseudouridimycin Biosynthesis

Abstract Pyridoxal 5′-phosphate (PLP)-dependent aminotransferases play a central role in nucleoside antibiotic biosynthesis, where they catalyze the conversion of ribose-derived aldehyde intermediates into aminonucleosides by introducing critical amino functional groups. Despite their widespread occurrence in the biosynthetic pathways of several therapeutically important nucleoside antibiotics, the detailed biochemical and mechanistic characterization of these enzymes remains elusive. In this study, we present a comprehensive in vitro reconstitution, structural modeling, and molecular dynamics (MD) simulation-based biochemical and mechanistic investigation of a key aminotransferase, PumG from Streptomyces rimosus (SrPumG), which plays a critical role in the biosynthesis of the potent bacterial RNA polymerase inhibitor, Pseudouridimycin (PUM). Our size-exclusion chromatography and native PAGE analysis, in conjunction with spectroscopic and modeling studies, showed that SrPumG is a stable homodimer that uses a highly conjugated form of the PLP cofactor, along with suitable amino donors, such as L-Arg, to convert pseudouridine aldehyde (PUA) to amino pseudouridine (APU). The structure-based three-dimensional modeling and MD simulation of SrPumG, coupled with site-directed mutagenesis, further confirmed that, in addition to the catalytic residue Lys289, several active-site residues from both protomers facilitate the binding and stabilization of the PLP/PMP cofactor and the PUA substrate via electrostatic/hydrogen-bonding interactions and aromatic stacking. In addition, our studies uncovered key residues that stabilize the dimer interface via hydrophobic and π–π interactions. One notable finding from our study is that SrPumG does not discriminate between C- and N-nucleoside substrates and exhibits a broader substrate scope, which further confirms the gatekeeper role of the preceding oxidoreductase enzyme, PumI, in this pathway. Together, these findings reveal that a distinct highly conjugated form of PLP and an intricate dimer interface architecture, which is essential for substrate/cofactor binding and protein stability, govern SrPumG catalysis. This work provides new insights into aminotransferases in nucleoside biosynthesis and establishes a foundation for engineering the PUM pathway to design new antibacterial derivatives.