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The existence of cobalamin (Cbl)-dependent enzymes that are members of the radical S-adenosyl-L-methionine (SAM) superfamily was previously predicted based on 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 sp3 carbon during the biosynthesis of gentamicin, an aminoglycoside antibiotic. Experiments to investigate the stoichiometry of the GenK reaction revealed that one equivalent 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. Based on 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.

A novel post‐translationally modified amino acid, crotonyllysine (Kcr), was genetically incorporated into proteins in bacterial and mammalian cells using an evolved pyrrolysyl‐tRNA/synthetase‐tRNA pair. The ability to produce histones with homogenous, site‐specific Kcr modifications will be valuable in elucidating the biological role of this recently identified post‐translational modification.

Spinosyn A is a polyketide-derived macrolide produced by Saccharopolyspora spinosa and is an active ingredient in several commercial insecticides. It is glycosylated by a tri-O-methylated rhamnose at C-9 and a forosamine at C-17. Previous studies indicated that the rhamnose methyltransferases are encoded by the spnH, spnI and spnK genes. To verify the functions of these methyltransferases and to study how they are coordinated to achieve the desired level of methylation of rhamnose, we studied the catalytic properties of the spnH, spnI and spnK gene products and validated their roles in the permethylation process of spinosyn A. Our data reported herein firmly established that SpnH, SpnI, and SpnK are the respective rhamnose 4′-, 2′-, and 3′-Omethyltransferase. Investigation of the order of the methylation events revealed that only one route catalyzed by SpnI, SpnK and SpnH in sequence is productive for the permethylation of the rhamnose moiety. Moreover, the completion of rhamnose permethylation is likely achieved by the proper control of the expression levels of the methyltransferase genes involved. These results set the stage for future exploitation of spinosyn biosynthetic pathway to produce targeted spinosyn derivatives and, perhaps, new analogues.Spinosyn A (SPA, 1) is a polyketide-derived macrolide produced by Saccharopolyspora spinosa, that is an active ingredient in several commercial insecticides. 1 The structures of SPA and its many analogues have a characteristic perhydro-as-indacene core, which is glycosylated by a rhamnose (see 2) at C-9 and a forosamine at C-17. Both the aglycone (AGL, 3) and the sugar appendages contribute to the observed activity of the spinosyns, among which SPA is most potent. 2 Alteration of the tetracyclic nucleus or removal of either deoxy sugars significantly diminishes the pesticidal activity. Even subtle structural variations, such as the methylation pattern of the rhamnose moiety in spinosyns, change the LD 50 by as much as >200- 3 the study of how the corresponding methyltransferases are coordinated to achieve the desired level of methylation of rhamnose has been a focus of this research. We report herein the function and substrate specificity of the three methyltransferases involved in the methylation reactions, the preferred reaction sequence of their catalyzed reactions, and the likely regulation of permethylation of rhamnose in 1.The spinosyn biosynthetic gene cluster had been cloned from S. spinosa. 4 Sequence analysis and gene disruption experiments revealed that the spnH, spnI and spnK genes, 4 all of which show high sequence identity to those encoding S-adenosyl-L-methionine (SAM) dependent methyltransferases (MTs), are involved in the O-methylation of rhamnose in 1. 4,5 As illustrated in Scheme 1, methylations may take place before (2 → 4 → 6, route A) or after (3 → 5 → 6, route B) the attachment of rhamnose to the aglycone (AGL, 3). It is also possible that methylations are the final tailoring steps after both sugars have been coupled to the aglycone (...

Abstract1-Deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase (DXR, also known as methyl-D-erythritol 4-phosphate (MEP) synthase) is a NADPH-dependent enzyme, which catalyzes the conversion of DXP to MEP in the non-mevalonate pathway of isoprene biosynthesis. Two mechanisms have been proposed for the DXR-catalyzed reaction. In the α-ketol rearrangement mechanism, the reaction begins with deprotonation of the C-3 hydroxyl group followed by a 1,2-migration to give methylerythrose phosphate, which is then reduced to MEP by NADPH. In the retroaldol/aldol rearrangement mechanism, DXR first cleaves the C3-C4 bond of DXP in a retroaldol manner to generate a three-carbon and a two-carbon phosphate bimolecular intermediate. These two species are then reunited by an aldol reaction to form a new C-C bond, yielding an aldehyde intermediate. Subsequent reduction by NADPH affords MEP. To differentiate these mechanisms, we have prepared [3-2 H]-and [4-2 H]-DXP and carried out a competitive secondary kinetic isotope effect (KIE) study of the DXR reaction. The normal 2° KIEs observed for [3-2 H]-and [4-2 H]-DXP provide compelling evidence supporting a retroaldol/aldol mechanism for the rearrangement catalyzed by DXR, with the rate-limiting step being cleavage of the C3-C4 bond of DXP.Terpenoids are a large family of secondary metabolites, consisting of more than 55,000 members, that are widely distributed in nature and rich in biological activities. 1,2 Terpenoids are biosynthesized starting with two 5-carbon isoprene units, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which have long been established to be derived from acetate in a pathway involving mevalonic acid as the key intermediate. 3 However, a new mevalonate-independent isoprene source has recently been discovered in eubacteria, archeabacteria, algae, and in the plastids of plants. [4][5][6] Since this pathway is absent in mammals but is essential for many pathogens, including Plasmodium falciparum 7 and Mycobacterium tuberculosis ,8 all enzymes in this pathway are potential antibacterial targets. 9The first committed step of this non-mevalonate pathway is the conversion of 1-deoxy-Dxylulose 5-phosphate (DXP, 1) to methyl-D-erythritol 4-phosphate (MEP, 2), catalyzed by the NADPH-dependent enzyme, DXP reductoisomerase (DXR, also known as MEP synthase, see Scheme 1). 10 Since MEP is the first metabolite specific to this pathway, this biosynthetic route is commonly referred to as the MEP pathway. Two mechanisms have been proposed for the Email: h.w.liu@mail.utexas.edu . DXR-catalyzed reaction (Scheme 1). In the α-ketol rearrangement mechanism (route A), the reaction begins with deprotonation of the C-3 hydroxyl group followed by a 1,2-(C4-to-C2)-migration to give methylerythrose phosphate (3), which is then reduced to MEP (2) by NADPH. NIH Public AccessIn the retroaldol/aldol mechanism (route B), DXR first cleaves the C3-C4 bond of 1 in a retroaldol manner to generate a three-carbon (4) and a two-carbon phosphate (5) Figure S1). 17 This phenomen...