![Author ORCID: We display the ORCID iD icon alongside authors names on our website to acknowledge that the ORCiD has been authenticated when entered by the user. To view the users ORCiD record click the icon. [opens in a new tab]](https://chemrxiv.org/engage/assets/public/chemrxiv/images/logos/orcid.png)
Abstract
Aminoglycosides are important antibiotics used to treat severe infections caused mainly by gram-negative bacteria. Gentamicin is an aminoglycoside, and despite its toxicity, is clinically used to treat several pulmonary and urinary infections. The commercial form of gentamicin is a mixture of five compounds with minor differences in the methylation of one of their aminosugars. In the case of two compounds, gentamicin C2 and C2a, the only difference is the stereochemistry of the methyl group attached to C-6’. GenB2 is the enzyme responsible for this epimerization and it is one of the four PLP-dependent enzymes encoded by the gentamicin biosynthetic gene cluster (BGC). Herein, we have determined the structure of GenB2 in its holo form in complex with PMP and also in the ternary complex with gentamicin X2 and G418, two substrate analogs. Based on the structural analysis, we were able to identify the structural basis for the catalytic mechanism of this enzyme, which was also studied by site-directed mutagenesis. Unprecedently, GenB2 is a PLP-dependent enzyme from fold I, which is able to catalyze an epimerization, but with a distinct mechanism from fold III PLP-dependent epimerases, using a cysteine residue near the N-terminus. The substitution of this cysteine residue for serine or alanine completely abolished the epimerase function of the enzyme confirming its involvement. This study not only contributes to the understanding of the enzymology of gentamicin biosynthesis but also provides valuable details for exploring the enzymatic production of new aminoglycoside derivatives.
Supplementary materials
Title
The structural and functional basis of GenB2 isomerase activity from gentamicin biosynthesis
Description
Supporting Information. Supplementary Table 1. Data collection and refinement statistics; Supplementary figure 1. Sequence alignment of different PLP-dependent enzymes involved in the biosynthesis of gentamicin and neomycin; Supplementary Figure 2. Residues involved in the dimerization of GenB2; Supplementary figure 3. Superposition of GenB2 with orthologous proteins; Supplementary figure 4. Electron density contours for the catalytic Lys227 and the coenzyme; Supplementary figure 5. Coenzyme (PMP) binding site conservation; Supplementary Figure 6. Substrate binding site of GenB2; Supplementary figure 7. Conservation of the binding mode of G418 (orange) and Gentamicin X2 (green) in the binding site of GenB2; Supplementary Figure 8. Superposition of GenB2 at different complexes; Supplementary figure 9. Binding mode of substrates and/or analogs on PLP-dependent enzymes from the biosynthesis of aminoglycosides.
Actions
