Biomedical subjects

Andrew L Lovering

Publications and source records attributed to Andrew L Lovering.

10 recordsLinked to original sources

PubMed2006

Structural analysis of an "open" form of PBP1B from Streptococcus pneumoniae.

The class A PBP1b from Streptococcus pneumoniae is responsible for glycosyltransferase and transpeptidase (TP) reactions, forming the peptidoglycan of the bacterial cell wall. The enzyme has been produced in a stable, soluble form and undergoes time-dependent proteolysis to leave an intact TP domain. Crystals of this TP domain were obtained, diffracting to 2.2 A resolution, and the structure was solved by using molecular replacement. Analysis of the structure revealed an "open" active site, with important conformational differences to the previously determined "closed" apoenzyme. The active-site nucleophile, Ser460, is in an orientation that allows for acylation by beta-lactams. Consistent with the productive conformation of the conserved active-site catalytic residues, adjacent loops show only minor deviation from those of known acyl-enzyme structures. These findings are discussed in the context of enzyme functionality and the possible conformational sampling of PBP1b between active and inactive states.

Bacterial Proteins↗

PubMed2006

Expanding the thioglycoligase strategy to the synthesis of alpha-linked thioglycosides allows structural investigation of the parent enzyme/substrate complex.

For the first time, the thioglycoligase strategy has been successfully applied to alpha-glycosidases. The alpha-thioglycoligases derived from the family 31 glycosidases, alpha-xylosidase from E. coli (YicI) and alpha-glucosidase from Sulfolobus solfataricus, catalyze thioglycoligase reactions using alpha-glycosyl fluorides and deoxythioglycosides as donors and acceptors, respectively, in yields up to 86%. In addition, we describe the Michaelis complex of YicI using one of the thioglycosides as a nonhydrolyzable substrate analogue and discuss the structural insights this yields into the specificity and mechanism of family 31 alpha-glycosidases and the molecular basis of an associated genetic disease.

Crystallography, X-Ray↗

PubMed2005

Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone. Reversed binding orientations in different redox states of the enzyme.

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 approximately 10,000-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.

Acetates↗

PubMed2005

Beta-lactam antibiotic resistance: a current structural perspective.

Bacterial resistance to beta-lactam antibiotics can be achieved by any of three strategies: the production of beta-lactam-hydrolyzing beta-lactamase enzymes, the utilization of beta-lactam-insensitive cell wall transpeptidases, and the active expulsion of beta-lactam molecules from Gram-negative cells by way of efflux pumps. In recent years, structural biology has contributed significantly to the understanding of these processes and should prove invaluable in the design of drugs to combat beta-lactam resistance in the future.

Anti-Bacterial Agents↗

PubMed2004

Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidis in complex with Mn2+, phosphoenolpyruvate, and N-acetylmannosaminitol.

In Neisseria meningitidis and related bacterial pathogens, sialic acids play critical roles in mammalian cell immunity evasion and are synthesized by a conserved enzymatic pathway that includes sialic acid synthase (NeuB, SiaC, or SynC). NeuB catalyzes the condensation of phosphoenolpyruvate (PEP) and N-acetylmannosamine, directly forming N-acetylneuraminic acid (or sialic acid). In this paper we report the development of a coupled assay to monitor NeuB reaction kinetics and an 18O-labeling study that demonstrates the synthase operates via a C-O bond cleavage mechanism. We also report the first structure of a sialic acid synthase, that of NeuB, revealing a unique domain-swapped homodimer architecture consisting of a (beta/alpha)8 barrel (TIM barrel)-type fold at the N-terminal end and a domain with high sequence identity and structural similarity to the ice binding type III antifreeze proteins at the C-terminal end of the enzyme. We have determined the structures of NeuB in the malate-bound form and with bound PEP and the substrate analog N-acetylmannosaminitol to 1.9 and 2.2 A resolution, respectively. Typical of other TIM barrel proteins, the active site of NeuB is located in a cavity at the C-terminal end of the barrel; however, the positioning of the swapped antifreeze-like domain from the adjacent monomer provides key residues for hydrogen bonding with substrates in the active site of NeuB, a structural feature that leads to distinct modes of substrate binding from other PEP-utilizing enzymes that lack an analogous antifreeze-like domain. Our observation of a direct interaction between a highly ordered manganese and the N-acetylmannosaminitol in the NeuB active site also suggests an essential role for the ion as an electrophilic catalyst that activates the N-acetylmannosamine carbonyl to the addition of PEP.

Amino Acid Sequence↗

PubMed2004

Mechanistic and structural analysis of a family 31 alpha-glycosidase and its glycosyl-enzyme intermediate.

We have determined the first structure of a family 31 alpha-glycosidase, that of YicI from Escherichia coli, both free and trapped as a 5-fluoroxylopyranosyl-enzyme intermediate via reaction with 5-fluoro-alpha-D-xylopyranosyl fluoride. Our 2.2-A 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 alpha-glucosidases lysosomal alpha-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.

Amino Acid Sequence↗

PubMed2004

Structural and biochemical identification of a novel bacterial oxidoreductase.

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.

Amino Acid Sequence↗

PubMed2004

Crystal structures of prostaglandin D(2) 11-ketoreductase (AKR1C3) in complex with the nonsteroidal anti-inflammatory drugs flufenamic acid and indomethacin.

It is becoming increasingly well established that nonsteroidal anti-inflammatory drugs (NSAID) protect against tumors of the gastrointestinal tract and that they may also protect against a variety of other tumors. These activities have been widely attributed to the inhibition of cylooxygenases (COX) and, in particular, COX-2. However, several observations have indicated that other targets may be involved. Besides targeting COX, certain NSAID also inhibit enzymes belonging to the aldo-keto reductase (AKR) family, including AKR1C3. We have demonstrated previously that overexpression of AKR1C3 acts to suppress cell differentiation and promote proliferation in myeloid cells. However, this enzyme has a broad tissue distribution and therefore represents a novel candidate for the target of the COX-independent antineoplastic actions of NSAID. Here we report on the X-ray crystal structures of AKR1C3 complexed with the NSAID indomethacin (1.8 A resolution) or flufenamic acid (1.7 A resolution). One molecule of indomethacin is bound in the active site, whereas flufenamic acid binds to both the active site and the beta-hairpin loop, at the opposite end of the central beta-barrel. Two other crystal structures (1.20 and 2.1 A resolution) show acetate bound in the active site occupying the proposed oxyanion hole. The data underline AKR1C3 as a COX-independent target for NSAID and will provide a structural basis for the future development of new cancer therapies with reduced COX-dependent side effects.

Amino Acid Sequence↗

PubMed2004

Nitroreductase: a prodrug-activating enzyme for cancer gene therapy.

1. The prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) is activated by Escherichia coli nitroreductase (NTR) to a potent DNA-crosslinking agent. 2. Virus-mediated expression of NTR in tumour cells sensitizes them to CB1954 in vitro and in vivo, providing the basis for a strategy of cancer gene therapy. 3. A phase I trial of CB1954 in cancer patients has been completed, documenting the pharmacokinetics and establishing an acceptable dose. Subsequent trials of the replication-defective adenovirus CTL102 in patients with resectable tumours have documented expression of NTR in injected colorectal liver metastases, hepatocellular carcinoma, head and neck cancer and prostate cancer. Trials combining CTL102 and CB1954 are underway. 4. An oncolytic (replication-competent) adenovirus vector allowed increased expression of NTR in vitro and in a mouse tumour model, resulting in a greater reduction in tumour growth when combined with CB1954 treatment. 5. Alternative prodrugs may eventually prove superior to CB1954; a nitroaryl phosphoramide mustard prodrug activated by NTR shows a greater therapeutic index than CB1954 in a human ovarian carcinoma. 6. The crystal structure of NTR provided the basis for site-directed mutagenesis, which has identified a number of mutants with improved kinetics of CB1954 activation. These can provide improved cell sensitization to CB1954. Combinations of these are being tested. 7. The basis for a positive selection for improved NTR variants has been demonstrated.

Animals↗

PubMed2003

Generation of Escherichia coli nitroreductase mutants conferring improved cell sensitization to the prodrug CB1954.

Escherichia coli nitroreductase (NTR) activates the prodrug CB1954 to a cytotoxic derivative, allowing selective sensitization of NTR-expressing cells or tumors to the prodrug. This is one of several enzyme-prodrug combinations that are under development for cancer gene therapy, and the system has now entered clinical trials. Enhancing the catalytic efficiency of NTR for CB1954 could improve its therapeutic potential. From the crystal structure of an enzyme-ligand complex, we identified nine amino acid residues within the active site that could directly influence prodrug binding and catalysis. Mutant libraries were generated for each of these residues and clones screened for their ability to sensitize E. coli to CB1954. Amino acid substitutions at six positions conferred markedly greater sensitivity to CB1954 than did the WT enzyme; the best mutants, at residue F124, resulted in approximately 5-fold improvement. Using an adenovirus vector, we introduced the F124K NTR mutant into human SK-OV-3 ovarian carcinoma cells and showed it to be approximately 5-fold more potent in sensitizing the cells to CB1954 at the clinically relevant prodrug concentration of 1 micro M than was the WT enzyme. Enhanced mutant NTRs such as F124K should improve the efficacy of the NTR/CB1954 combination in cancer gene therapy.

Adenoviridae↗