GenK-catalyzed C-6' methylation in the biosynthesis of gentamicin: isolation and characterization of a cobalamin-dependent radical SAM enzyme

Abstract

The existence of cobalamin (Cbl)-dependent enzymes that are members of the radical S-adenosyl-l-methionine (SAM) superfamily was previously predicted on the basis of bioinformatic analysis. A number of these are Cbl-dependent methyltransferases, but the details surrounding their reaction mechanisms have remained unclear. In this report we demonstrate the in vitro activity of GenK, a Cbl-dependent radical SAM enzyme that methylates an unactivated sp(3) carbon during the biosynthesis of gentamicin, an aminoglycoside antibiotic. Experiments to investigate the stoichiometry of the GenK reaction revealed that 1 equiv each of 5'-deoxyadenosine and S-adenosyl-homocysteine are produced for each methylation reaction catalyzed by GenK. Furthermore, isotope-labeling experiments demonstrate that the S-methyl group from SAM is transferred to Cbl and the aminoglycoside product during the course of the reaction. On the basis of these results, one mechanistic possibility for the GenK reaction can be ruled out, and further questions regarding the mechanisms of Cbl-dependent radical SAM methyltransferases, in general, are discussed.

Figure 1

HPLC traces showing the production of both 5′-dAdo (4) and SAH (5) during the GenK reaction. Trace (a) is a standard composed of authentic 5′-dAdo (4) and SAH (5). The remaining traces result from reaction mixtures prepared as described in the text containing (b) MeCbl, (c) HOCbl, (d) MeCbl but no GenK, (e) HOCbl but no GenK, (f) MeCbl but no GenX₂, (g) no Cbl, (h) MeCbl and non-reconstituted GenK. Detector was set at 260 nm.

Figure 2

HPLC traces showing conversion of GenX₂ (7) to G418 (8) in the presence of GenK. Prior to HPLC analysis, the assay mixture was subjected to the reaction with 1-fluoro-2,4-dinitrobenzene (DNFB) (see SI for details). Absorbance was monitored at 340 nm to detect FDNB-derivatized aminoglycosides. Trace (a) is a standard composed of derivatized, authentic GenX₂ (7) and G418 (8). Traces b–h are as described in the legend of Fig. 1. The small peaks at ~16.6 min in traces d–g and ~15.4 min in trace f are unrelated to product and substrate.

Figure 3

Mass spectra of aminoglycoside substrate and product displaying incorporation of ¹³CD₃ from ¹³CD₃-methyl-SAM into product. The spectra correspond to assays with (a) HOCbl + unlabeled SAM, (b) HOCbl + ¹³CD₃-methyl-SAM, (c) HOCbl + unlabeled SAM without GenK, (d) HOCbl + ¹³CD₃-methyl-SAM without GenK, (e) MeCbl + unlabeled SAM, (f) MeCbl + ¹³CD₃-methyl-SAM, (g) MeCbl + unlabeled SAM without GenK, (h) MeCbl + ¹³CD₃-methyl-SAM without GenK.

Figure 4

Stoichiometry of 5′-dAdo and SAH production during the GenK reaction.

Scheme 1

Initial reaction catalyzed by radical SAM enzymes.

Scheme 2

The C-6′ methylation reaction catalyzed by GenK in the gentamicin biosynthetic pathway.

Scheme 3

Possible GenK reaction mechanisms. (A) GenX₂ radical (9) is quenched by the transfer of methyl radical from Me-Cbl(III) to give Cbl(II), which is reduced to Cbl(I) in order to accept a new methyl group from SAM. (B) Transfer of methyl cation to GenX₂ ketyl radical (11) followed by reduction and protonation of product radical (12). (C) Product radical (12) is quenched by a hydrogen from 5′-dAdo (4) to give 5′-dAdo• (3), which can be re-incorporated into SAM. In this mechanism, a single equivalent of SAM can serve as a source of both 5′-dAdo• (3) and CH₃⁺.