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.

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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...

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.

Carbohydrates are highly abundant biomolecules found extensively in nature. Besides playing important roles in energy storage and supply, they often serve as essential biosynthetic precursors or structural elements needed to sustain all forms of life. A number of unusual sugars that have certain hydroxyl groups replaced by a hydrogen, an amino group, or an alkyl side chain play crucial roles in determining the biological activity of the parent natural products in bacterial lipopolysaccharides or secondary metabolite antibiotics. Recent investigation of the biosynthesis of these monosaccharides has led to the identification of the gene clusters whose protein products facilitate the unusual sugar formation from the ubiquitous NDP-glucose precursors. This review summarizes the mechanistic studies of a few enzymes crucial to the biosynthesis of C-2, C-3, C-4, and C-6 deoxysugars, the characterization and mutagenesis of nucleotidyl transferases that can recognize and couple structural analogs of their natural substrates and the identification of glycosyltransferases with promiscuous substrate specificity. Information gleaned from these studies has allowed pathway engineering, resulting in the creation of new macrolides with unnatural deoxysugar moieties for biological activity screening. This represents a significant progress toward our goal of searching for more potent agents against infectious diseases and malignant tumors.

D-Desosamine (1) is a 3-(N,N-dimethylamino)-3,4,6-trideoxyhexose found in a number of macrolide antibiotics including methymycin (2), neomethymycin (3), pikromycin (4), and narbomycin (5) produced by Streptomyces venezuelae. It plays an essential role in conferring biological activities to its parent aglycones. Previous genetic and biochemical studies of the biosynthesis of desosamine in S. venezuelae showed that the conversion of TDP-4-amino-4,6-dideoxy-D-glucose (8) to TDP-3-keto-4,6-dideoxy-D-glucose (9) is catalyzed by DesII, which is a member of the radical S-adenosyl-L-methionine (SAM) enzyme superfamily. Here, we report the purification and reconstitution of His 6 -tagged DesII, characterization of its [4Fe-4S] cluster using UV-Vis and EPR spectroscopies, and the capability of flavodoxin, flavodoxin reductase, and NADPH, to reduce the [4Fe-4S] 2+ cluster. Also included are a steady-state kinetic analysis of DesII-catalyzed reaction and an investigation of the substrate flexibility of DesII. Studies of deuterium incorporation into SAM using TDP-[3-2 H]-4-amino-4,6-dideoxy-D-glucose as the substrate provides strong evidence for direct hydrogen atom transfer to a 5′-deoxyadenosyl radical in the catalytic cycle. The fact that hydrogen atom abstraction occurs at C-3 also sheds light on the mechanism of this intriguing deamination reaction. D-Desosamine (1) is a 3-(N,N-dimethylamino)-3,4,6-trideoxyhexose found in a number of macrolide antibiotics including methymycin (2), neomethymycin (3), narbomycin (4), and pikromycin (5) produced by Streptomyces venezuelae. Both biochemical and structural studies have shown that desosamine is an essential structural component crucial to the biological activity of the parent aglycones (e.g., 12, 13). 1 Desosamine is biosynthetically derived from TDP-D-glucose (6), and a key step in its formation is the removal of the C-4 hydroxyl group of the hexose ring. Early studies of desosamine biosynthesis in S. venezuelae revealed that the C-4 deoxygenation is unique among biological deoxygenation processes 2 and the reaction proceeds in two stages: the conversion of TDP-4-keto-6-deoxy-D-glucose (7) to the corresponding 4-amino sugar intermediate (8), and the deamination of 8 to afford TDP-3-keto-4,6-dideoxy-D-glucose as the final product (9 , Scheme 1). The former reaction is catalyzed by DesI, a pyridoxal-5′-phosphate (PLP)-dependent aminotransferase, whereas the latter reaction is catalyzed by DesII, a radical S-adenosyl-L-methionine (SAM)-dependent enzyme. 3 Subsequent C-3 aminotransfer (9→ 10) by DesV followed by N,N-dimethylation (10→ 11) by DesVI complete the desosamine biosynthesis. 4 All enzymes in the pathway have been biochemically characterized, but the details of the catalytic properties of DesII and the mechanism of its catalysis remain obscure.*To whom correspondence should be addressed. Fax: 512-471-2746. h.w.liu@mail.utexas NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThus far, more than 2800 proteins have been identified, mainly b...

It has been shown that 1-aminocyclopropane-1-carboxylic acid (ACC) is the immediate precursor of ethylene, which is derived from C-2 and C-3 of ACC. When [1-14C]ACC was administered to etiolated mungbean (Vigna radiata) hypocotyls, -16% of the ACC was converted to ethylene and about 10% of the radioactivity was converted to[14C]asparagine in 7 hr. In etiolated epicotyls of common vetch (Vicia saliva), after 7 hr about 14% of the ACC was converted to ethylene and 16% of the radioactivity was converted to 1 cyanoalanine plus -glutamyl--cyanoalanine. It is known that in most plants cyanide is metabolized to asparagine via the intermediate (3cyanoalanine, whereas in a few plants such as V. saliva, 1-cyanoalanine is converted to the conjugate y-glutamyl-f8-cyanoalanine. We