Characterization of SpnQ from the Spinosyn Biosynthetic Pathway of Saccharopolyspora spinosa:  Mechanistic and Evolutionary Implications for C-3 Deoxygenation in Deoxysugar Biosynthesis

Abstract

The C-3 deoxygenation step in the biosynthesis of D-forosamine (4-N,N-dimethylamino-2,3,4,6-tetradeoxy-D-threo-hexopyranose), a constituent of spinosyn produced by Saccharopolyspora spinosa, was investigated. The spnQ gene, proposed to encode a TDP-4-keto-2,6-dideoxy-D-glucose 3-dehydratase was cloned and overexpressed in E. coli. Characterization of the purified enzyme established that it is a PMP and iron-sulfur containing enzyme which catalyzes the C-3 deoxygenation in a reductase-dependent manner similar to that of the previously well characterized hexose 3-dehydrase E 1 from Yersinia pseudotuberculosis. However, unlike E 1 , which has evolved to work with a specific reductase partner present in its gene cluster, SpnQ lacks a specific reductase, and works efficiently with general cellular reductases ferredoxin/ferredoxin reductase or flavodoxin/ flavodoxin reductase. SpnQ also catalyzes C-4 transamination in the absence of an electron transfer intermediary and in the presence of PLP and L-glutamate. Under the same conditions, both E 1 and the related hexose 3-dehydrase, ColD, catalyze C-3 deoxygenation. Thus, SpnQ possesses important features which distinguish it from other well studied homologues, suggesting unique evolutionary pathways for each of the three hexose 3-dehydrases studied thus far.As part of our effort to characterize unusual sugar biosynthetic pathways and to collect genes useful for in vivo glycodiversification of secondary metabolites, 1 we investigated the biosynthesis of D-forosamine (1), a key structural component of the spinosyns, which are produced by Saccharopolyspora spinosa 2a and Saccharopolyspora pogona. 2b Spinosad, a mixture of Spinosyn A and D (2, 3 , Scheme 1A), is a potent yet environmentally benign insecticide. 2a Structure-activity studies have shown that D-forosamine is essential for the insecticidal properties of the spinosyns. 3 There is also evidence that Spinosad promotes wound healing in humans. 4The Spinosyn (spn) biosynthetic gene cluster has been cloned and sequenced, and the genes spnO, spnN, spnQ, spnR, spnS, and spnP were proposed to be involved in forosamine biosynthesis and attachment. 2a In a recent study, SpnR was shown to be a PLP-dependent aminotransferase catalyzing the conversion of 7 to 8. 5 It is thus suggested that forosamine formation is initiated by C-2 deoxygenation, and likely proceeds via a sequence of (4 → 5 → 6 → 7 → 8 , Scheme 1A). C-3 deoxygenation (6 → 7) is proposed to be mediated by SpnQ, which shows modest sequence identity to pyridoxamine 5′-monophosphate (PMP)-dependent

Characterization of the Amicetin Biosynthesis Gene Cluster from Streptomyces vinaceusdrappus NRRL 2363 Implicates Two Alternative Strategies for Amide Bond Formation

“…Thus, AmiD may reduce the C-3 keto in compound 10 to form the equatorial hydroxyl group in TDP-4-keto-2,6-dideoxy-D-glucose (compound 11). The subsequent C-3 deoxygenation of compound 11, leading to compound 12, is probably performed by AmiN in a way similar to that of its homologue SpnQ (72% identity), a mechanistically characterized TDP-4-keto-6-deoxyglucose-3-dehydratase in the spinosyn pathway (34). Finally, the C-4 reduction on compound 12 to form TDP-Damicetose (compound 13) is putatively catalyzed by AmiK, which is homologous to a predicted NDP-hexose 4-ketoreductase, PokS6 (35% identity), from the polyketomycin pathway (15).…”

Natural‐Product Sugar Biosynthesis and Enzymatic Glycodiversification

“…The biosynthesis of TDP-d-forosamine (100) in the spinosyn-producing strain, Saccharopolyspora spinosa, has been fully elucidated in vitro. In this work, SpnQ was shown to be the 3-dehydrase converting 73 to 91, [112] and SpnR was identified as the 4-transaminase converting the SpnQ product 91 to TDP-4-amino-2,3,4,6-tetradeoxy-d-glucose (99). [113] The N,N-dimethylation of 99 is catalyzed by SpnS.…”

“…Interestingly, unlike its homologue E 1 (see Section 3.1.5 for a mechanistic discussion), SpnQ does not have a dedicated reductase partner encoded in the gene cluster, but it instead uses general cellular reductases such as ferredoxin and flavodoxin for electron transfer. [112][113][114] …”

The spinosyn family of insecticides: realizing the potential of natural products research

“…51 Other recent studies have further characterized the enzymes involved in biosynthesis of the amino-and neutral sugars. [52][53][54][55] The currently favored biosynthetic pathway for converting the aglycone (7) to spinosyn A (1) proceeds by addition of rhamnose, sequential 2¢-, 3¢-and 4¢-O-methylation and addition of forosamine ( Figure 5). Further studies exploring the entire pathway of spinosyn biosynthesis and genetic engineering of various stages are likely to continue and to uncover intriguing results and novel applications.…”