Methyltransferase Contingencies in the Pathway of Everninomicin D Antibiotics and Analogues.

Everninomicins are orthoester oligosaccharide antibiotics with potent activity against multidrug-resistant bacterial pathogens. Everninomicins act by disrupting ribosomal assembly in a distinct region in comparison to clinically prescribed drugs. We employed microporous intergeneric conjugation with Escherichia coli to manipulate Micromonospora for targeted gene-replacement studies of multiple putative methyltransferases across the octasaccharide scaffold of everninomicin effecting the A1 , C, F, and H rings. Analyses of gene-replacement and genetic complementation mutants established the mutability of the everninomicin scaffold through the generation of 12 previously unreported analogues and, together with previous results, permitted assignment of the ten methyltransferases required for everninomicin biosynthesis. The in vitro activity of A1 - and H-ring-modifying methyltransferases demonstrated the ability to catalyze late-stage modification of the scaffold on an A1 -ring phenol and H-ring C-4' hydroxy moiety. Together these results establish the potential of the everninomicin scaffold for modification through mutagenesis and in vitro modification of advanced biosynthetic intermediates.

Bifunctional Nitrone-Conjugated Secondary Metabolite Targeting the Ribosome.

Many microorganisms possess the capacity for producing multiple antibiotic secondary metabolites. In a few notable cases, combinations of secondary metabolites produced by the same organism are used in important combination therapies for treatment of drug-resistant bacterial infections. However, examples of conjoined roles of bioactive metabolites produced by the same organism remain uncommon. During our genetic functional analysis of oxidase-encoding genes in the everninomicin producer Micromonospora carbonacea var. aurantiaca, we discovered previously uncharacterized antibiotics everninomicin N and O, comprised of an everninomicin fragment conjugated to the macrolide rosamicin via a rare nitrone moiety. These metabolites were determined to be hydrolysis products of everninomicin P, a nitrone-linked conjugate likely the result of nonenzymatic condensation of the rosamicin aldehyde and the octasaccharide everninomicin F, possessing a hydroxylamino sugar moiety. Rosamicin binds the erythromycin macrolide binding site approximately 60 Å from the orthosomycin binding site of everninomicins. However, while individual ribosomal binding sites for each functional half of everninomicin P are too distant for bidentate binding, ligand displacement studies demonstrated that everninomicin P competes with rosamicin for ribosomal binding. Chemical protection studies and structural analysis of everninomicin P revealed that everninomicin P occupies both the macrolide- and orthosomycin-binding sites on the 70S ribosome. Moreover, resistance mutations within each binding site were overcome by the inhibition of the opposite functional antibiotic moiety binding site. These data together demonstrate a strategy for coupling orthogonal antibiotic pharmacophores, a surprising tolerance for substantial covalent modification of each antibiotic, and a potential beneficial strategy to combat antibiotic resistance.

An alternative N-terminal fold of the intestine-specific annexin A13a induces dimerization and regulates membrane-binding.

Annexin proteins function as Ca2+-dependent regulators of membrane trafficking and repair that may also modulate membrane curvature. Here, using high-resolution confocal imaging, we report that the intestine-specific annexin A13 (ANX A13) localizes to the tips of intestinal microvilli and determined the crystal structure of the ANX A13a isoform to 2.6 Å resolution. The structure revealed that the N terminus exhibits an alternative fold that converts the first two helices and the associated helix-loop-helix motif into a continuous α-helix, as stabilized by a domain-swapped dimer. We also found that the dimer is present in solution and partially occludes the membrane-binding surfaces of annexin, suggesting that dimerization may function as a means for regulating membrane binding. Accordingly, as revealed by in vitro binding and cellular localization assays, ANX A13a variants that favor a monomeric state exhibited increased membrane association relative to variants that favor the dimeric form. Together, our findings support a mechanism for how the association of the ANX A13a isoform with the membrane is regulated.

The Structure of the Bifunctional Everninomicin Biosynthetic Enzyme EvdMO1 Suggests Independent Activity of the Fused Methyltransferase-Oxidase Domains.

Members of the orthosomycin family of natural products are decorated polysaccharides with potent antibiotic activity and complex biosynthetic pathways. The defining feature of the orthosomycins is an orthoester linkage between carbohydrate moieties that is necessary for antibiotic activity and is likely formed by a family of conserved oxygenases. Everninomicins are octasaccharide orthosomycins produced by Micromonospora carbonacea that have two orthoester linkages and a methylenedioxy bridge, three features whose formation logically requires oxidative chemistry. Correspondingly, the evd gene cluster encoding everninomicin D encodes two monofunctional nonheme iron, α-ketoglutarate-dependent oxygenases and one bifunctional enzyme with an N-terminal methyltransferase domain and a C-terminal oxygenase domain. To investigate whether the activities of these domains are linked in the bifunctional enzyme EvdMO1, we determined the structure of the N-terminal methyltransferase domain to 1.1 Å and that of the full-length protein to 3.35 Å resolution. Both domains of EvdMO1 adopt the canonical folds of their respective superfamilies and are connected by a short linker. Each domain's active site is oriented such that it faces away from the other domain, and there is no evidence of a channel connecting the two. Our results support EvdMO1 working as a bifunctional enzyme with independent catalytic activities.

Structural Basis for Sialoglycan Binding by the Streptococcus sanguinis SrpA Adhesin.

Streptococcus sanguinisis a leading cause of infective endocarditis, a life-threatening infection of the cardiovascular system. An important interaction in the pathogenesis of infective endocarditis is attachment of the organisms to host platelets.S. sanguinisexpresses a serine-rich repeat adhesin, SrpA, similar in sequence to platelet-binding adhesins associated with increased virulence in this disease. In this study, we determined the first crystal structure of the putative binding region of SrpA (SrpABR) both unliganded and in complex with a synthetic disaccharide ligand at 1.8 and 2.0 Å resolution, respectively. We identified a conserved Thr-Arg motif that orients the sialic acid moiety and is required for binding to platelet monolayers. Furthermore, we propose that sequence insertions in closely related family members contribute to the modulation of structural and functional properties, including the quaternary structure, the tertiary structure, and the ligand-binding site.

Oxidative cyclizations in orthosomycin biosynthesis expand the known chemistry of an oxygenase superfamily.

Orthosomycins are oligosaccharide antibiotics that include avilamycin, everninomicin, and hygromycin B and are hallmarked by a rigidifying interglycosidic spirocyclic ortho-δ-lactone (orthoester) linkage between at least one pair of carbohydrates. A subset of orthosomycins additionally contain a carbohydrate capped by a methylenedioxy bridge. The orthoester linkage is necessary for antibiotic activity but rarely observed in natural products. Orthoester linkage and methylenedioxy bridge biosynthesis require similar oxidative cyclizations adjacent to a sugar ring. We have identified a conserved group of nonheme iron, α-ketoglutarate-dependent oxygenases likely responsible for this chemistry. High-resolution crystal structures of the EvdO1 and EvdO2 oxygenases of everninomicin biosynthesis, the AviO1 oxygenase of avilamycin biosynthesis, and HygX of hygromycin B biosynthesis show how these enzymes accommodate large substrates, a challenge that requires a variation in metal coordination in HygX. Excitingly, the ternary complex of HygX with cosubstrate α-ketoglutarate and putative product hygromycin B identified an orientation of one glycosidic linkage of hygromycin B consistent with metal-catalyzed hydrogen atom abstraction from substrate. These structural results are complemented by gene disruption of the oxygenases evdO1 and evdMO1 from the everninomicin biosynthetic cluster, which demonstrate that functional oxygenase activity is critical for antibiotic production. Our data therefore support a role for these enzymes in the production of key features of the orthosomycin antibiotics.

Structure determination and characterization of the vitamin B6 degradative enzyme (E)-2-(acetamidomethylene)succinate hydrolase.

The gene identification and kinetic characterization of (E)-2-(acetamidomethylene)succinate (E-2AMS) hydrolase has recently been described. This enzyme catalyzes the final reaction in the degradation of vitamin B(6) and produces succinic semialdehyde, acetate, ammonia, and carbon dioxide from E-2AMS. The structure of E-2AMS hydrolase was determined to 2.3 A using SAD phasing. E-2AMS hydrolase is a member of the alpha/beta hydrolase superfamily and utilizes a serine/histidine/aspartic acid catalytic triad. Mutation of either the nucleophilic serine or the aspartate resulted in inactive enzyme. Mutation of an additional serine residue in the active site causes the enzyme to be unstable and is likely structurally important. The structure also provides insight into the mechanism of hydrolysis of E-2AMS and identifies several potential catalytically important residues.

Structure of the PLP degradative enzyme 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase from Mesorhizobium loti MAFF303099 and its mechanistic implications.

A vitamin B(6) degradative pathway has recently been identified and characterized in Mesorhizobium loti MAFF303099. One of the enzymes on this pathway, 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO), is a flavin-dependent enzyme and catalyzes the oxidative ring-opening of 2-methyl-3-hydroxypyridine-5-carboxylic acid to form E-2-(acetamino-methylene)succinate. The gene for this enzyme has been cloned, and the corresponding protein has been overexpressed in Escherichia coli and purified. The crystal structure of MHPCO has been solved to 2.1 A using SAD phasing with and without the substrate MHPC bound. These crystal structures provide insight into the reaction mechanism and suggest roles for active site residues in the catalysis of a novel oxidative ring-opening reaction.

Structural studies of thiamin monophosphate kinase in complex with substrates and products.

Thiamin monophosphate kinase (ThiL) catalyzes the ATP-dependent phosphorylation of thiamin monophosphate (TMP) to form thiamin pyrophosphate (TPP), the active form of vitamin B 1. ThiL is a member of a small ATP binding superfamily that also includes the purine biosynthetic enzymes, PurM and PurL, NiFe hydrogenase maturation protein, HypE, and selenophosphate synthase, SelD. The latter four enzymes are believed to utilize phosphorylated intermediates during catalysis. To understand the mechanism of ThiL and its relationship to the other superfamily members, we determined the structure of Aquifex aeolicus ThiL (AaThiL) with nonhydrolyzable AMP-PCP and TMP, and also with the products of the reaction, ADP and TPP. The results suggest that AaThiL utilizes a direct, inline transfer of the gamma-phosphate of ATP to TMP rather than a phosphorylated enzyme intermediate. The structure of ThiL is compared to those of PurM, PurL, and HypE, and the ATP binding site is compared to that of PurL, for which nucleotide complexes are available.

Gene identification and structural characterization of the pyridoxal 5'-phosphate degradative protein 3-hydroxy-2-methylpyridine-4,5-dicarboxylate decarboxylase from mesorhizobium loti MAFF303099.

The function of the mlr6791 gene from Mesorhizobium loti MAFF303099 has been identified. This gene encodes 3-hydroxy-2-methylpyridine-4,5-dicarboxylate decarboxylase (HMPDdc), an enzyme involved in the catabolism of pyridoxal 5'-phosphate (Vitamin B6). This enzyme was overexpressed in Escherichia coli and characterized. HMPDdc is a 26 kDa protein that catalyzes the decarboxylation of 3-hydroxy-2-methylpyridine-4,5-dicarboxylate to 3-hydroxy-2-methylpyridine-5-carboxylate. The KM and kcat were found to be 366 microM and 0.6 s-1, respectively. The structure of this enzyme was determined at 1.9 A resolution using SAD phasing and belongs to the class II aldolase/adducin superfamily. While the decarboxylation of hydroxy-substituted benzene rings is a common motif in biosynthesis, the mechanism of this reaction is still poorly characterized. The structural studies described here suggest that catalysis of such decarboxylations proceeds by an aldolase-like mechanism.

Identification of a domain in the V0 subunit d that is critical for coupling of the yeast vacuolar proton-translocating ATPase.

Vacuolar proton-translocating ATPase pumps consist of two domains, V(1) and V(o). Subunit d is a component of V(o) located in a central stalk that rotates during catalysis. By generating mutations, we showed that subunit d couples ATP hydrolysis and proton transport. The mutation F94A strongly uncoupled the enzyme, preventing proton transport but not ATPase activity. C-terminal mutations changed coupling as well; ATPase activity was decreased by 59-72%, whereas proton transport was not measurable (E328A) or was moderately reduced (E317A and C329A). Except for W325A, which had low levels of V(1)V(o), mutations allowed wild-type assembly regardless of the fact that subunits E and d were reduced at the membrane. N- and C-terminal deletions of various lengths were inhibitory and gradually destabilized subunit d, limiting V(1)V(o) formation. Both N and C terminus were required for V(o) assembly. The N-terminal truncation 2-19Delta prevented V(1)V(o) formation, although subunit d was available. The C terminus was required for retention of subunits E and d at the membrane. In addition, the C terminus of its bacterial homolog (subunit C from T. thermophilus) stabilized the yeast subunit d mutant 310-345Delta and allowed assembly of the rotor structure with subunits A and B. Structural features conserved between bacterial and eukaryotic subunit d and the significance of domain 3 for vacuolar proton-translocating ATPase function are discussed.