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.

The recently identified type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase (IDI-2) is a flavoenzyme that requires FMN and NAD(P)H for activity. IDI-2 is an essential enzyme for the biosynthesis of isoprenoids in several pathogenic bacteria including Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis, and thus is considered as a potential new drug target to battle bacterial infections. One notable feature of the IDI-2 reaction is that there is no net change in redox state between the substrate (IPP) and product (DMAPP), indicating that the FMN cofactor must start and finish each catalytic cycle in the same redox state. Here, we report the characterization and initial mechanistic studies of the S. aureus IDI-2. The steady-state kinetic analyses under aerobic and anaerobic conditions show that FMN must be reduced to be catalytically active and the overall IDI-2 reaction is O2-sensitive. Interestingly, our results demonstrate that NADPH is needed only in catalytic amounts to activate the enzyme for multiple turnovers of IPP to DMAPP. The hydride transfer from NAD(P)H to reduce FMN is determined to be pro-S stereospecific. Photoreduction and oxidation-reduction potential studies reveal that the S. aureus IDI-2 can stabilize significant amounts of the neutral FMN semiquinone. In addition, reconstitution of apo-IDI-2 with 5-deazaFMN resulted in a dead enzyme, whereas reconstitution with 1-deazaFMN led to the full recovery of enzyme activity. Taken together, these studies appear to support a catalytic mechanism in which the reduced flavin coenzyme mediates a single electron transfer to and from the IPP substrate during catalysis.

Deoxysugars are critical structural elements for the bioactivity of many natural products. Ongoing work on elucidating a variety of deoxysugar biosynthetic pathways has paved the way for manipulation of these pathways for the generation of structurally diverse glycosylated natural products. In the course of this work, the biosynthesis of d-mycaminose in the tylosin pathway of Streptomyces fradiae was investigated. Attempts to reconstitute the entire mycaminose biosynthetic machinery in a heterologous host led to the discovery of a previously overlooked gene, tyl1a, encoding an enzyme thought to convert TDP-4-keto-6-deoxy-d-glucose to TDP-3-keto-6-deoxy-d-glucose, a 3,4-ketoisomerization reaction in the pathway. Tyl1a has now been overexpressed, purified, and assayed, and its activity has been verified by product analysis. Incubation of Tyl1a and the C-3 aminotransferase TylB, the next enzyme in the pathway, produced TDP-3-amino-3,6-dideoxy-d-glucose, confirming that these two enzymes act sequentially. Steady state kinetic parameters of the Tyl1a-catalyzed reaction were determined, and the ability of Tyl1a and TylB to process a C-2 deoxygenated substrate and a CDP-linked substrate was also demonstrated. Enzymes catalyzing 3,4-ketoisomerization of hexoses represent a new class of enzymes involved in unusual sugar biosynthesis. The fact that Tyl1a exhibits a relaxed substrate specificity holds potential for future deoxysugar biosynthetic engineering endeavors.

One goal of synthetic chemistry is to prepare a target molecule by a minimum number of transformations with high stereo-selectivity and efficiency. Despite significant advances made in recent years in the field of synthetic chemistry, preparation of many organic compounds still require multi-step chemical reactions. The lengthy operations can be tedious and timeconsuming, and can suffer from low yields. However, as more biosynthetic pathways for natural products are characterized, the idea of exploiting these pathways to produce desired products has become increasingly attractive. It has recently been demonstrated that the biosynthetic machineries can be manipulated to produce tailor-made new compounds. 1 Moreover, due to the high regio-and stereo-specificity of enzyme catalysis, the enzymatic synthesis may be carried out in one-pot, 2 which is not possible for most chemical reactions. Such an approach avoids the isolation and/or the accumulation of unstable intermediates, and also minimizes the use of chemicals and production of waste. The environmentally benign nature of these biosynthetic approaches is highly attractive.As a part of our effort to elucidate the biosynthetic pathways of unusual sugars, we have recently cloned and identified a number of sugar biosynthetic gene clusters, 3 including the entire pathway for the formation of mycarose (1). 4 L-Mycarose is a key component of the macrolide antibiotic tylosin (2), which is produced by Streptomyces fradiae. It is also found in a few other clinically useful antibiotics. In order to generate new bioactive compounds by derivatization of structurally diverse macrolide aglycones with L-mycarose using selected glycosyltransferases, it is necessay to have large quantities of TDP-L-mycarose (3) readily accessible as it is the sugar substrate form recognized by the glycosyltransferases. Although L-mycarose has already been chemically synthesized, 5 attempts to prepare TDP-L-mycarose are hampered by the facile loss of the TDP substituent at C-1. To circumvent this problem, we explored a biosynthetic approach for the preparation of the compound.As shown in Scheme 1, TDP-L-mycarose (3) is derived from TDP-D-glucose (4) via six enzymatic reactions. 4 The key intermediate 7 is produced from 4 in three separate steps catalyzed by a 4,6-dehydratase (TylA2, 4 → 5), 4 a 2-dehydrase (TylX3, 5 → 6), 6 and a 3-reductase (TylC1, 6 → 7). 6 Subsequently, 7 is transformed to 8 by methylation at C-3 by a methyltransferase (TylC3), 7 8 is converted to 9 by epimerization at C-5 by an epimerase (TylK), 4 and the reduction of 9 at C-4 by a reductase (TylC2) 4 yields TDP-L-mycarose (3). Two constraints make a one-pot synthesis of 3 a necessity. First, compound 6 is inherently unstable since incubation of 5 with TylX3 results in its rapid consumption with the concomitant formation of TDP and maltol. 7,8 Thus, the inclusion of TylC1 and NADPH in the reaction is required to reduce the labile 6 in situ to form the more stable product 7. Second, the in situ reduction catalyzed by TylC2 is ...