In the biosynthetic pathway of the spinosyn insecticides, the tailoring enzyme SpnF performs a [4+2]-cycloaddition on a 22-membered macrolactone to forge an embedded cyclohexene ring. To learn more about this reaction, which could potentially proceed through a Diels-Alder mechanism, the 1.50 Å-resolution crystal structure of SpnF bound to S-adenosylhomocysteine (SAH) was determined. This sets the stage for advanced experimental and computational studies to determine the precise mechanism of SpnF-mediated cyclization.

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 (...

Spinosyns A and D (spinosad) are complex polyketide natural products biosynthesized through the cooperation of a modular polyketide synthase and several tailoring enzymes. SpnP catalyzes the final tailoring step, transferring forosamine from a TDP-d-forosamine donor substrate to a spinosyn pseudoaglycone acceptor substrate. Sequence analysis indicated that SpnP belongs to a small group of glycosyltransferases (GTs) that require an auxiliary protein for activation. However, unlike other GTs in this subgroup, no putative auxiliary protein gene could be located in the biosynthetic gene cluster. To learn more about SpnP, the structures of SpnP and its complex with TDP were determined to 2.50 and 3.15 Å resolution, respectively. Binding of TDP causes the reordering of several residues in the donor substrate pocket. SpnP possesses a structural feature that has only been previously observed in the related glycosyltransferase EryCIII, in which it mediates association with the auxiliary protein EryCII. This motif, H-X-R-X5-D-X5-R-X12–20-D-P-X3-W-L-X12–18-E-X4-G, may be predictive of glycosyltransferases that interact with an auxiliary protein. A reverse glycosyl transfer assay demonstrated that SpnP possesses measurable activity in the absence of an auxiliary protein. Our data suggest that SpnP can bind its donor substrate by itself but that the glycosyl transfer reaction is facilitated by an auxiliary protein that aids in the correct folding of a flexible loop surrounding the pseudoaglycone acceptor substrate-binding pocket.

Spinosyns A and D (spinosad), like many other complex polyketides, are tailored near the end of their biosyntheses through the addition of sugars. SpnG, which catalyzes their 9-OH rhamnosylation, is also capable of adding other monosaccharides to the spinosyn aglycone (AGL) from TDP-sugars; however, the substitution of UDP-d-glucose for TDP-d-glucose as the donor substrate is known to result in a >60,000-fold reduction in kcat. Here, we report the structure of SpnG at 1.65 Å-resolution, SpnG bound to TDP at 1.86 Å-resolution, and SpnG bound to AGL at 1.70 Å-resolution. The SpnG-TDP complex reveals how SpnG employs N202 to discriminate between TDP- and UDP-sugars. A conformational change of several residues in the active site is promoted through the binding of TDP. The SpnG-AGL complex shows that the binding of AGL is mediated through hydrophobic interactions and that H13, the potential catalytic base, is within 3 Å of the nucleophilic AGL 9-OH. A model for the Michaelis complex was constructed to reveal the features that enable SpnG to transfer diverse sugars; it also revealed that the rhamnosyl moiety is in a skew-boat conformation during the transfer reaction.

Translation is a tightly regulated process that ensures optimal protein quality and enables adaptation to energy/nutrient availability. The α-kinase eukaryotic elongation factor 2 kinase (eEF-2K), a key regulator of translation, specifically phosphorylates the guanosine triphosphatase eEF-2, thereby reducing its affinity for the ribosome and suppressing the elongation phase of protein synthesis. eEF-2K activation requires calmodulin binding and autophosphorylation at the primary stimulatory site, T348. Biochemical studies predict a calmodulin-mediated activation mechanism for eEF-2K distinct from other calmodulin-dependent kinases. Here, we resolve the atomic details of this mechanism through a 2.3-Å crystal structure of the heterodimeric complex of calmodulin and the functional core of eEF-2K (eEF-2KTR). This structure, which represents the activated T348-phosphorylated state of eEF-2KTR, highlights an intimate association of the kinase with the calmodulin C-lobe, creating an “activation spine” that connects its amino-terminal calmodulin-targeting motif to its active site through a conserved regulatory element.