Many biologically active small-molecule natural products produced by microorganisms derive their activities from sugar substituents. Changing the structures of these sugars can have a profound impact on the biological properties of the parent compounds. This realization has inspired attempts to derivatize the sugar moieties of these natural products through exploitation of the sugar biosynthetic machinery. This approach requires an understanding of the biosynthetic pathway of each target sugar and detailed mechanistic knowledge of the key enzymes. Scientists have begun to unravel the biosynthetic logic behind the assembly of many glycosylated natural products and have found that a core set of enzyme activities is mixed and matched to synthesize the diverse sugar structures observed in nature. Remarkably, many of these sugar biosynthetic enzymes and glycosyltransferases also exhibit relaxed substrate specificity. The promiscuity of these enzymes has prompted efforts to modify the sugar structures and alter the glycosylation patterns of natural products through metabolic pathway engineering and enzymatic glycodiversification. In applied biomedical research, these studies will enable the development of new glycosylation tools and generate novel glycoforms of secondary metabolites with useful biological activity.

The antibiotic kijanimicin produced by the actinomycete Actinomadura kijaniata has a broad spectrum of bioactivities as well as a number of interesting biosynthetic features. To understand the molecular basis for its formation and to develop a combinatorial biosynthetic system for this class of compounds, a 107.6 kb segment of the Actinomadura kijaniata chromosome containing the kijanimicin biosynthetic locus was identified, cloned, and sequenced. The complete pathway for the formation of TDP-L-digitoxose, one of the two sugar donors used in construction of kijanimicin, was elucidated through biochemical analysis of four enzymes encoded in the gene cluster. Sequence analysis indicates that the aglycone kijanolide is formed by the combined action of a modular Type-I polyketide synthase (PKS) and a conserved operon involved attachment and intramolecular cyclization of a glycerate-derived three-carbon unit, which forms the core of the spirotetronate moiety. The genes involved in the biosynthesis of the unusual deoxysugar D-kijanose [2,3,4,6-tetradeoxy-4-(methylcarbamyl)-3-C-methyl-3-nitro-D-xylo-hexopyranose], including one encoding a flavoenzyme predicted to catalyze the formation of the nitro group, have also been identified. This work has implications for the biosynthesis of other spirotetronate antibiotics and nitro sugar-bearing natural products, as well as for future mechanistic and biosynthetic engineering efforts.Kijanimicin (1) is a spirotetronate antibiotic isolated from Actinomadura kijaniata, a soil actinomycete. It has a broad spectrum of antimicrobial activity against Gram-positive bacteria, anaerobes, and the malaria parasite Plasmodium falciparum, 1 and also shows antitumor activity. 2 The structure of kijanimicin (1) consists of a pentacyclic core, which is equipped with four L-digitoxose (2) units and a rare nitro sugar, 2,3,4,6-tetradeoxy-4-(methylcarbamyl)-3-C-methyl-3-nitro-D-xylo-hexopyranose, commonly known as D-kijanose (3). More than sixty kijanimicin-related spirotetronate-type compounds have been reported. Most are made by strains of high-GC Gram positive bacteria (Actinomycetes), including Streptomyces, 3-8 Micromonospora, 9-12 Actinomadura, 1,13,14 Saccharothrix, 15 and Verrucosispora. 16 A species of Bacillus has also been identified as a producer of a member of this class of compounds. 17 Nearly all members of this class exhibit both antibacterial and antitumor activities, and many possess other biological activities. Well-known examples include chlorothricins (4), the anticholesterolemic agents; 18,19 tetronothiodin, a cholecystokinin B (CCK-B) inhibitor; 4 MM46115, an antiviral drug effective against parainfluenzae virus 1 and 2; 13 and tetrocarcins (5) and arisostatins, both of which have been shown to have therapeutic potential as inducers of apoptosis. 20-23 In a recent study, a collection of tetrocarcin analogues was prepared synthetically and some of them showed improved apoptosis-inducing activity. 24 Hence, compounds of this class have broad therapeutic potential wort...

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SpnF, an enzyme involved in the biosynthesis of spinosyn A, catalyzes a transannular Diels–Alder reaction. Quantum mechanical computations and dynamic simulations now show that this cycloaddition is not well described as either a concerted or stepwise process, and dynamical effects influence the identity and timing of bond formation. The transition state for the reaction is ambimodal and leads directly to both the observed Diels–Alder and an unobserved [6+4] cycloadduct. The potential energy surface bifurcates and the cycloadditions occur by dynamically stepwise modes featuring an “entropic intermediate”. A rapid Cope rearrangement converts the [6+4] adduct into the observed [4+2] adduct. Control of nonstatistical dynamical effects may serve as another way by which enzymes control reactions.

The biosynthetic pathway of the clinically important antibiotic fosfomycin uses enzymes that catalyse reactions without precedent in biology. Among these is hydroxypropylphosphonic acid epoxidase, which represents a new subfamily of non-haem mononuclear iron enzymes. Here we present six X-ray structures of this enzyme: the apoenzyme at 2.0 A resolution; a native Fe(II)-bound form at 2.4 A resolution; a tris(hydroxymethyl)aminomethane-Co(II)-enzyme complex structure at 1.8 A resolution; a substrate-Co(II)-enzyme complex structure at 2.5 A resolution; and two substrate-Fe(II)-enzyme complexes at 2.1 and 2.3 A resolution. These structural data lead us to suggest how this enzyme is able to recognize and respond to its substrate with a conformational change that protects the radical-based intermediates formed during catalysis. Comparisons with other family members suggest why substrate binding is able to prime iron for dioxygen binding in the absence of alpha-ketoglutarate (a co-substrate required by many mononuclear iron enzymes), and how the unique epoxidation reaction of hydroxypropylphosphonic acid epoxidase may occur.