Structural and\nFunctional Basis of GenB2 Isomerase\nActivity from Gentamicin Biosynthesis

Aminoglycosides are\nessential antibiotics used to treat severe\ninfections caused mainly by Gram-negative bacteria. Gentamicin is\nan aminoglycoside and, despite its toxicity, is clinically used to\ntreat several pulmonary and urinary infections. The commercial form\nof gentamicin is a mixture of five compounds with minor differences\nin the methylation of one of their aminosugars. In the case of two\ncompounds, gentamicin C2 and C2a, the only difference is the stereochemistry\nof the methyl group attached to C-6′. GenB2 is the enzyme responsible\nfor this epimerization and is one of the four PLP-dependent enzymes\nencoded by the gentamicin biosynthetic gene cluster. Herein, we have\ndetermined the structure of GenB2 in its holo form in complex with\nPMP and also in the ternary complex with gentamicin X2 and G418, two\nsubstrate analogues. Based on the structural analysis, we were able\nto identify the structural basis for the catalytic mechanism of this\nenzyme, which was also studied by site-directed mutagenesis. Unprecedently,\nGenB2 is a PLP-dependent enzyme from fold I, which is able to catalyze\nan epimerization but with a mechanism distinct from that of fold III\nPLP-dependent epimerases using a cysteine residue near the N-terminus.\nThe substitution of this cysteine residue for serine or alanine completely\nabolished the epimerase function of the enzyme, confirming its involvement.\nThis study not only contributes to the understanding of the enzymology\nof gentamicin biosynthesis but also provides valuable details for\nexploring the enzymatic production of new aminoglycoside derivatives.