Specificity and Promiscuity at the Branch Point in Gentamicin Biosynthesis

Abstract

SummaryGentamicin C complex is a mixture of aminoglycoside antibiotics used to treat severe Gram-negative bacterial infections. We report here key features of the late-stage biosynthesis of gentamicins. We show that the intermediate gentamicin X2, a known substrate for C-methylation at C-6′ to form G418 catalyzed by the radical SAM-dependent enzyme GenK, may instead undergo oxidation at C-6′ to form an aldehyde, catalyzed by the flavin-linked dehydrogenase GenQ. Surprisingly, GenQ acts in both branches of the pathway, likewise oxidizing G418 to an analogous ketone. Amination of these intermediates, catalyzed mainly by aminotransferase GenB1, produces the known intermediates JI-20A and JI-20B, respectively. Other pyridoxal phosphate-dependent enzymes (GenB3 and GenB4) act in enigmatic dehydroxylation steps that convert JI-20A and JI-20B into the gentamicin C complex or (GenB2) catalyze the epimerization of gentamicin C2a into gentamicin C2.

Virtual Force Term Based on the Velocity Distribution Surrounding a Spherical Particle Settling in a Simple Shear Flow

“…Δ gen B2 produced C1a and C2a along with ZL101, whereas C1 and C2 were undetectable (Figure 1 E). Mutant strains Δ gen B1, Δ gen B2, and Δ gen B2Δ gen B1 all produced C1a; this was consistent with a recent study on the functions of GenB1 and GenB2 in the gentamicin biosynthetic pathway,11 but we did not delve into the functions of GenB1 and GenB2 in gentamicin C1a biosynthesis.…”

“…Compound G418 is first converted into JI‐20B, with the same 6′‐chiral carbon as is present in gentamicins C2a and C2 11. In this study, as expected, in‐frame deletion within gen P, blocking the gentamicin pathway at JI‐20A and JI‐20B, resulted in the generation of the pair of epimers JI‐20Ba and JI‐20B (Scheme ).…”

“…The conversion of G418 to JI‐20B (and JI‐20Ba) is performed in a two‐step oxidation/transamination process. GenQ is a flavin‐dependent dehydrogenase that oxidizes G418 to 6′‐dehydro‐6′‐oxo‐G418 11. Purified recombinant GenQ, GenB1, and GenB2 proteins (Figure S11) were used for enzymatic reactions in vitro.…”

“…Gentamicin X2 and G418 are converted into JI‐20A and JI‐20B, respectively, through coupled oxidation/transamination reactions. GenQ catalyzes the oxidation of X2 and G418 11. 12 GenP catalyzes the 3′‐OH phosphorylation step of JI‐20A, which is possibly the first step for C‐3′ dehydroxylation 13…”

“…Recently, four potential aminotransferase‐encoding genes— gen B1, gen B2, gen B3, and gen B4—in the gentamicin gene cluster have been reported 11. In the formation of JI‐20A and JI‐20B, transamination is mainly catalyzed by GenB1, whereas GenB2 catalyzes the epimerization between C2a and C2; C2a here is the first product of JI‐20B bisdehydroxylation.…”