Peptidoglycan glycosyltransferases (GTs) catalyze the polymerization step of cell-wall biosynthesis, are membrane-bound, and are highly conserved across all bacteria. Long considered the "holy grail" of antibiotic research, they represent an essential and easily accessible drug target for antibiotic-resistant bacteria, including methicillin-resistant Staphylococcus aureus. We have determined the 2.8 angstrom structure of a bifunctional cell-wall cross-linking enzyme, including its transpeptidase and GT domains, both unliganded and complexed with the substrate analog moenomycin. The peptidoglycan GTs adopt a fold distinct from those of other GT classes. The structures give insight into critical features of the catalytic mechanism and key interactions required for enzyme inhibition.

The peptidoglycan biosynthetic pathway is a critical process in the bacterial cell and is exploited as a target for the design of antibiotics. This pathway culminates in the production of the peptidoglycan layer, which is composed of polymerized glycan chains with cross-linked peptide substituents. This layer forms the major structural component of the protective barrier known as the cell wall. Disruption in the assembly of the peptidoglycan layer causes a weakened cell wall and subsequent bacterial lysis. With bacteria responsible for both properly functioning human health (probiotic strains) and potentially serious illness (pathogenic strains), a delicate balance is necessary during clinical intervention. Recent research has furthered our understanding of the precise molecular structures, mechanisms of action, and functional interactions involved in peptidoglycan biosynthesis. This research is helping guide our understanding of how to capitalize on peptidoglycan-based therapeutics and, at a more fundamental level, of the complex machinery that creates this critical barrier for bacterial survival.

The antibiotics nitrofurazone and nitrofurantoin are used in the treatment of genitourinary infections and as topical antibacterial agents. Their action is dependent upon activation by bacterial nitroreductase flavoproteins, including the Escherichia coli nitroreductase (NTR). Here we show that the products of reduction of these antibiotics by NTR are the hydroxylamine derivatives. We show that the reduction of nitrosoaromatics is enzyme-catalyzed, with a specificity constant ϳ10,000-fold greater than that of the starting nitro compounds. This suggests that the reduction of nitro groups proceeds through two successive, enzyme-mediated reactions and explains why the nitroso intermediates are not observed. The global reaction rate for nitrofurazone determined in this study is over 10-fold higher than that previously reported, suggesting that the enzyme is much more active toward nitroaromatics than previously estimated. Surprisingly, in the crystal structure of the oxidized NTR-nitrofurazone complex, nitrofurazone is oriented with its amide group, rather than the nitro group to be reduced, positioned over the reactive N5 of the FMN cofactor. Free acetate, which acts as a competitive inhibitor with respect to NADH, binds in a similar orientation. We infer that the orientation of bound nitrofurazone depends upon the redox state of the enzyme. We propose that the charge distribution on the FMN rings, which alters upon reduction, is an important determinant of substrate binding and reactivity in flavoproteins with broad substrate specificity.

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We have determined the first structure of a family 31 ␣-glycosidase, that of YicI from Escherichia coli, both free and trapped as a 5-fluoroxylopyranosyl-enzyme intermediate via reaction with 5-fluoro-␣-D-xylopyranosyl fluoride. Our 2.2-Å resolution structure shows an intimately associated hexamer with structural elements from several monomers converging at each of the six active sites. Our kinetic and mass spectrometry analyses verified several of the features observed in our structural data, including a covalent linkage from the carboxylate side chain of the identified nucleophile Asp 416 to C-1 of the sugar ring. Structure-based sequence comparison of YicI with the mammalian ␣-glucosidases lysosomal ␣-glucosidase and sucrase-isomaltase predicts a high level of structural similarity and provides a foundation for understanding the various mutations of these enzymes that elicit human disease.Glycoside hydrolases play critical roles in biology ranging from digestion and decomposition of polysaccharides to biosynthesis of glycoproteins. Gene sequences of many thousands of these important enzymes have now been determined, and the corresponding enzymes have been grouped into families on the basis of sequence similarity (1-5). 1The ␣-glucosidases are a particularly important subset of these enzymes, both in primary metabolism and in glycoconjugate biosynthesis and processing. These enzymes are principally found in families 13 and 31 and, to a lesser extent, in families 4 and 63. Family 13 contains a wide range of glucosideprocessing enzymes, including the ␣-amylases and cyclodextrin glucanotransferases. Correspondingly, enzymes of this family have attracted considerable attention, with numerous mechanistic studies and with three-dimensional structures having been known for some 20 years. By contrast, family 31 has received relatively little attention despite its importance and the number of different activities represented from a range of organisms, including animals, plants, and microorganisms (6, 7). This family contains such important ␣-glucosidases as the human lysosomal ␣-glucosidase, a deficiency of which results in Pompe's disease (also known as glycogen storage disease type 2 or acid maltase deficiency, the marked feature of which is the accumulation of glycogen in heart and skeletal muscle cells); the endoplasmic reticulum glucosidase II, which plays a key role in glycoprotein processing and folding; and the digestive enzyme sucrase-isomaltase, which is the target of inhibition by the anti-diabetes drugs acarbose and miglitol. It also contains ␣-xylosidases, isomaltosyltransferases, and the mechanistically interesting ␣-glucan lyases, which carry out an elimination reaction rather than hydrolysis.Despite the importance of this family, mechanistic insights are limited. The enzymes are known to be retaining ␣-glycosidases, which hydrolyze the glycosidic bond with net retention of anomeric configuration via an acid/base-catalyzed mechanism involving a covalent glycosyl-enzyme intermediate. Through a range of st...

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By using a bioinformatics screen of the Escherichia coli genome for potential molybdenum-containing enzymes, we have identified a novel oxidoreductase conserved in the majority of Gram-negative bacteria. The identified operon encodes for a proposed heterodimer, YedYZ in Escherichia coli, consisting of a soluble catalytic subunit termed YedY, which is likely anchored to the membrane by a heme-containing trans-membrane subunit termed YedZ. YedY is uniquely characterized by the presence of one molybdenum molybdopterin not conjugated by an additional nucleotide, and it represents the only molybdoenzyme isolated from E. coli characterized by the presence of this cofactor form. We have further characterized the catalytic subunit YedY in both the molybdenum-and tungsten-substituted forms by using crystallographic analysis. YedY is very distinct in overall architecture from all known bacterial reductases but does show some similarity with the catalytic domain of the eukaryotic chicken liver sulfite oxidase. However, the strictly conserved residues involved in the metal coordination sphere and in the substrate binding pocket of YedY are strikingly different from that of chicken liver sulfite oxidase, suggesting a catalytic activity more in keeping with a reductase than that of a sulfite oxidase. Preliminary kinetic analysis of YedY with a variety of substrates supports our proposal that YedY and its many orthologues may represent a new type of membrane-associated bacterial reductase.Molybdenum-coordinating enzymes fall within the broad class of enzymes associated with redox metabolic functions in prokaryotic and eukaryotic cells. The structurally characterized enzymes can be roughly grouped into three separate families (the bacterial/eukaryotic xanthine oxidase family, the eukaryotic sulfite oxidase family, and the bacterial Me 2 SO reductase family), each distinctive with respect to active site structure and the type of reaction they catalyze (1). The family of xanthine oxidases contains 1 eq of a pterin cofactor coordinated to the molybdenum metal with the typical pentavalent, approximately octahedral coordination sphere in the oxidized state completed not by any side chains from the enzyme but rather by a double-bonded sulfur atom, a double-bonded oxygen atom, and an oxygen atom with a single bond (2). Sulfite oxidases have 1 eq of a pterin cofactor with the molybdenum coordinated by a cysteine ligand from the enzyme and two oxo groups (3, 4). Kappler et al. (5) described the spectroscopic and enzymologic characterization of a member of the sulfite oxidase family from Thiobacillus novellus, and they showed that the enzyme contains a molybdopterin-type cofactor, but no structural data are available for bacterial sulfite oxidase family members to verify the nature of the cofactor or overall architecture of this enzyme. The Me 2 SO reductase family is diverse in both structure and function, but all members have 2 eq of the pterin cofactor, and the molybdenum coordination sphere is usually completed by a single oxo group ...

The Mla pathway is believed to be involved in maintaining the asymmetrical Gramnegative outer membrane via retrograde phospholipid transport. The pathway is composed of 3 components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here we solve the crystal structure of MlaC in its phospholipid free closed apo conformation, revealing a pivoting βsheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the inner membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provides evidence of an apparatus for lipid export away from the inner membrane and suggests that the Mla pathway may have a role in anterograde phospholipid transport.