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G D Wright

Publications and source records attributed to G D Wright.

At least 73 records · Page 4Linked to original sources
PubMed1996

Catalytic mechanism of enterococcal kanamycin kinase (APH(3')-IIIa): viscosity, thio, and solvent isotope effects support a Theorell-Chance mechanism.

Bacterial resistance to the aminoglycoside antibiotics is manifested primarily through the production of enzymes which covalently modify these drugs. The Enterococci and Staphylococci produce an ATP-dependent kinase, APH(3')-IIIa, which phosphorylates such antibiotics as kanamycin, amikacin, and neomycin, and this enzyme shows a Theorell-Chance kinetic mechanism by traditional product and analogue inhibitor analysis and by the alternative substrate diagnostic [McKay, G. A., & Wright, G. D. (1995) J. Biol. Chem. 270, 24686-24692]. We report that the APH(3')-IIIa exhibits small solvent (VH/VD approximately equal to 1.50) and thio effects (VATP/VATP gamma S = 2) indicating hydroxyl group deprotonation and nucleophilic attack on ATP do not significantly contribute to the overall steady-state rate. The enzymatic rates were determined with the viscogens PEG 8000, glycerol, and sucrose, and these experiments demonstrate that ATP binding and ADP release are diffusion controlled and that ADP release is solely rate limiting for APH(3')-IIIa. In addition, the slope of V/K for ATP vs relative viscosity is greater than the theoretical limit of 1, suggesting a possible enzyme conformational change upon binding of ATP. This new experimental evidence supports a Theorell-Chance mechanism for APH(3')-IIIa.

Adenosine Diphosphate↗
PubMed1996

Regiospecificity of aminoglycoside phosphotransferase from Enterococci and Staphylococci (APH(3')-IIIa).

The broad-spectrum aminoglycoside phosphotransferase, APH(3')-IIIa, confers resistance to several aminoglycoside antibiotics in opportunistic pathogens of the genera Staphylococcus and Enterococcus. The profile of the drug resistance phenotype suggested that the enzyme would transfer a phosphate group from ATP to the 3'-hydroxyl of aminoglycosides. In addition, resistance to the 3'-deoxyaminoglycoside antibiotic, lividomycin A, suggested possible transfer to the 5"-hydroxyl of the ribose [Trieu-Cuot, P., & Courvalin, P. (1983) Gene 23, 331-341]. Using purified overexpressed enzyme, we have prepared and purified the products of APH(3')-IIIa-dependent phosphorylation of several of aminoglycoside antibiotics. Mass spectral analysis revealed that 4,6-disubstituted aminocyclitol antibiotics such as amikacin and kanamycin are monophosphorylated, while 4,5-disubstituted aminoglycosides such as butirosin A, ribostamycin, and neomycin B are both mono- and diphosphorylated by APH(3')-IIIa. Using a series of one- and two-dimensional 1H, 13C, and 31P NMR experiments, we have unambiguously assigned the regiospecificity of phosphoryl transfer to several antibiotics. The 4,6-disubstituted aminocyclitol antibiotics are exclusively phosphorylated at the 3'-OH hydroxyl, and the 4,5-disubstituted aminocyclitol antibiotics can be phosphorylated at both the 3'- and 5"-hydroxyls. The first phosphorylation can occur on either the 3'- or 5"-hydroxyl group of neomycin B or butirosin A. Initial phosphotransfer to the 3'-position predominates for butirosin while the 5"-OH is favored for neomycin. These results open the potential for the rational design of aminoglycoside kinase inhibitors based on functionalization of either the 6-aminohexose or the pentose rings of aminoglycoside antibiotics.

Aminoglycosides↗
PubMed1996

Purification and characterization of two haloperoxidases from the glycopeptide antibiotic producer Streptomyces toyocaensis NRRL 15009.

Streptomyces toyocaensis NRRL 15009 produces A47934, a glycopeptide antibiotic. This compound is composed of several unusual amino acids, some of which have chlorinated aromatic rings. We have isolated two distinct halogenating enzymes, a chloroperoxidase (42,882 Da) and a catalase/bromoperoxidase (53,890 Da), from late log phase drug producing cultures of this organism grown on soy based media. Both these enzymes are azide sensitive and show absorption spectra consistent with the presence of an iron-heme group. We have characterized these enzymes with respect to substrate specificity, steady state kinetics, molecular mass and N-terminal sequence. The catalase/bromoperoxidase is similar to an enzyme from the chloramphenicol producer, Streptomyces venezuelae, while the chloroperoxidase is a unique protein.

Amino Acid Sequence↗
PubMed1996

Memory for objects presented early after intracarotid sodium amytal: a sensitive clinical neuropsychological indicator of temporal lobe pathology.

Results from a simple test of post-recovery recognition of objects presented immediately after intracarotid sodium amytal (ISA) injection were compared with those obtained using the 'Montreal' anterograde memory test procedure of post-recovery recognition of items presented later after injection in 16 patients with unilateral temporal lobe pathology undergoing routine bilateral ISA testing prior to epilepsy surgery. All 16 patients were given both memory tests following injection on both sides. Significantly fewer 'early objects' were recognized when injection was contralateral to pathology than when injection was ipsilateral to pathology (i.e. contralateral to an intact hemisphere), whereas there was no significant difference in the number of 'Montreal' anterograde items recognized regardless of side of pathology. Memory for objects presented early after ISA appears to be a sensitive measure although its potential as a valid indicator of temporal lobe pathology needs to be further refined.

Adolescent↗
PubMed1996

Mechanism of aminoglycoside 3'-phosphotransferase type IIIa: His188 is not a phosphate-accepting residue.

BACKGROUND: The enzyme aminoglycoside 3'-phosphotransferase Type IIIa (APH(3')-IIIa), confers resistance to many aminoglycoside antibiotics by regiospecific phosphorylation of their hydroxyl groups. The chemical mechanism of phosphoryl transfer is unknown. Based on sequence homology, it has been suggested that a conserved His residue, His188, could be phosphorylated by ATP, and this phospho-His would transfer the phosphate to the incoming aminoglycoside. We have used chemical modification, site-directed mutagenesis and positional isotope exchange methods to probe the mechanism of phosphoryl transfer by APH(3')-IIIa. RESULTS: Chemical modification by diethylpyrocarbonate implicated His in aminoglycoside phosphorylation by APH(3')-IIIa. We prepared His --> Ala mutants of all four His residues in APH(3')-IIIa and found minimal effects of the mutations on the steady-state phosphorylation of several aminoglycosides. One of these mutants, His188Ala, was largely insoluble when compared to the wild-type enzyme. Positional isotope exchange experiments using gamma-[18O]-ATP did not support a double-displacement mechanism. CONCLUSIONS: His residues are not required for aminoglycoside phosphorylation by APH(3')-IIIa. The conserved His 188 is thus not a phosphate accepting residue but does seem to be important for proper enzyme folding. Positional isotope exchange experiments are consistent with direct attack of the aminoglycoside hydroxyl group on the gamma-phosphate of ATP.

Adenosine Triphosphate↗
PubMed1996

Recognition of aminoglycoside antibiotics by enterococcal-staphylococcal aminoglycoside 3'-phosphotransferase type IIIa: role of substrate amino groups.

The interactions of the aminoglycoside 3'-phosphotransferase IIIa with aminoglycoside antibiotics lacking specific amino groups were examined by steady-state kinetic analyses. The results demonstrate that an amino group on C-1 and either an amino or a hydroxyl group at the 2' and 6' positions are important for detoxification of aminoglycosides by this enzyme.

Amines↗
PubMed1996

Association of two loci on chromosome 2q with nodal osteoarthritis.

OBJECTIVE: To search for genetic association between microsatellite marker loci and sibling pairs with nodal osteoarthritis (NOA). METHODS: Using the affected sibling pair method of analysis, genomic DNA from 66 sib pairs with NOA was analysed for association with highly polymorphic microsatellite marker loci. The microsatellite markers were amplified using polymerase chain reaction and typed on polyacrylamide gels. RESULTS: A significant association (p < 0.05) was identified between NOA and two loci on the short arm of chromosome 2 (2q 23-35). Candidate genes for osteoarthritis in this region include: fibronectin, a glycoprotein present in the extracellular matrix of normal cartilage; the alpha 2 chain of collagen type V, a major constituent of bone; and the interleukin-8 receptor, important in the regulation of neutrophil activation and chemotaxis. CONCLUSIONS: The chromosomal region 2q 23-35 requires further detailed study in NOA. Confirmation of these findings in large independent data sets and further analysis of candidate genes in this region will be important in unravelling the molecular basis for this common disease.

Adult↗
PubMed1995

Kinetic mechanism of aminoglycoside phosphotransferase type IIIa. Evidence for a Theorell-Chance mechanism.

Bacterial resistance to aminoglycoside-aminocyclitol antibiotics is mediated primarily by covalent modification of the drugs by a variety of enzymes. One such modifying enzyme, the 3'-aminoglycoside phosphotransferase, which is produced by Gram-positive cocci such as Enterococcus and Streptococcus inactivates a broad range of aminoglycosides by ATP-dependent phosphorylation of specific hydroxyl residues on the antibiotics. Through the use of dead-end and product inhibitor studies, we present the first detailed examination of the kinetic mechanism for the 3'-aminoglycoside phosphotransferase-IIIa. Initial velocity patterns deduced from steady-state kinetics indicate a sequential mechanism with ordered binding of ATP first followed by aminoglycoside. Dead-end inhibition by AMP and adenylyl-imidodiphosphate is competitive versus ATP and noncompetitive versus kanamycin A. Dead-end inhibition by tobramycin, a kanamycin analogue lacking a 3'-OH, is competitive versus both kanamycin A and uncompetitive versus ATP, indicative of ordered substrate binding where ATP must add prior to aminoglycoside addition. Product inhibition by kanamycin phosphate is noncompetitive versus ATP when kanamycin A is held at subsaturating concentrations (Km(kanA)), whereas no inhibition is observed when the concentration of kanamycin A is held at 10Km(kanA). This is consistent with kanamycin phosphate being the first product released followed by ADP release. The patterns of inhibition observed support a mechanism where ATP binding precedes aminoglycoside binding followed by a rapid catalytic step. Product release proceeds in an ordered fashion where kanamycin phosphate is released quickly followed by a slow release of ADP. Aminoglycoside substrates, such as kanamycin A, show substrate inhibition that is uncompetitive versus ATP. This indicates binding of the aminoglycosides to the slowly dissociating (E-ADP) complex at high drug concentrations. These experiments are consistent with a Theorell-Chance kinetic mechanism for 3'-aminoglycoside phosphotransferase-IIIa.

Adenosine Triphosphate↗
PubMed1995

Overexpression, purification, and characterization of VanX, a D-, D-dipeptidase which is essential for vancomycin resistance in Enterococcus faecium BM4147.

Vancomycin resistance in Enterococcus faecium requires five genes: vanR, vanS, vanH, vanA, and vanX. The functions and mechanism of four gene products have been known, with VanR/S for signal transduction and transcriptional regulation and VanH/A to synthesize D-Ala-D-lactate. But the function of the fifth gene product, VanX, has been unknown until very recently, when Reynolds and colleagues discovered D-, D-dipeptidase activity in crude extracts of a VanX overproducer [Reynolds, P. E., et al. (1994) Mol. Microbiol. 13, 1065-1070]. We report here the expression of VanX in Escherichia coli and its purification to homogeneity. VanX has been characterized as a metal-activated D-, D-dipeptidase with an optimal pH range of 7-9. The kcat and Km of D-Ala-D-Ala in the absence of divalent metal are determined to be 4.7 s-1 and 1 mM, respectively. However, in the presence of metal cations, kcat can be as high as 788 s-1. VanX is unable to hydrolyze D-Ala-D-lactate, the substituted moiety in the peptidoglycan that leads to vancomycin resistance, not only because of low binding affinity (Ki estimated at 242 mM) but also due to a kcat less than 0.005 s-1. The more than 10(5)-fold differential in catalytic efficiency of VanX for hydrolysis of D-Ala-D-Ala vs D-Ala-D-lactate leaves D-Ala-D-lactate intact for subsequent incorporation into peptidoglycan.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Active-site labeling of an aminoglycoside antibiotic phosphotransferase (APH(3')-IIIa).

The aminoglycoside antibiotics are inactivated by modifying enzymes that are now widely distributed in many pathogenic bacteria. This situation threatens the continued use of these clinically important drugs. We have undertaken studies to understand the molecular mechanism of aminoglycoside resistance, and we report the affinity labeling of the enterococcal aminoglycoside 3'-phosphotransferase, APH(3')-IIIa, with an electrophilic ATP analogue, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA). Incubation of purified APH(3')-IIIa with FSBA resulted in time-dependent irreversible inactivation of enzyme activity with a binding constant, Ki, of 0.406 mM and a rate of maximal inactivation, kmax, of 0.086 min-1. Addition of ATP completely protected the enzyme from inactivation, consistent with labeling of the ATP binding site. Reaction of APH(3')-IIIa with [14C]FSBA showed that inactivated APH(3')-IIIa incorporates 1 mol of FSBA/mol of enzyme. Peptide mapping of FSBA-inactivated APH(3')-IIIa resulted in the identification of two peptide peaks with highly increased absorbance at 260 nm, indicative of covalent labeling with FSBA. Analysis by electrospray ionization mass spectrometry and Edman degradation revealed two tryptic peptides, Val31-Lys44 and Leu34-Arg49, which incorporated the FSBA label at Lys33 and Lys44, respectively. This establishes the importance of the N-terminal region of APHs in ATP binding, a region of these enzymes which has heretofore not been considered for involvement in substrate binding.

Adenosine↗
PubMed1994

Broad spectrum aminoglycoside phosphotransferase type III from Enterococcus: overexpression, purification, and substrate specificity.

The aminoglycoside phosphotransferases (APHs) are responsible for the bacterial inactivation of many clinically useful aminoglycoside antibiotics. We report the characterization of an enterococcal enzyme, APH(3')-IIIa, which inactivates a broad spectrum of aminoglycosides by ATP-dependent O-phosphorylation. Overproduction of APH(3')-IIIa has permitted the isolation of 30-40 mg of pure protein/(L of cell culture). Purified APH(3')-IIIa is a mixture of monomer and dimer which is slowly converted to dimer only over time. Dimer could be dissociated into monomer by incubation with 2-mercaptoethanol, suggesting that dimerization is mediated by formation of disulfide bond(s). Both monomer and dimer show Km values in the low micromolar range for good substrates such as kanamycin and neomycin, and kcat values of 1-4 s-1. All aminoglycosides show substrate inhibition except amikacin and kanamycin B. Determination of minimum inhibitory concentrations indicates a positive correlation between antibiotic activity and kcat/Km, but not with Km or kcat. NMR analysis of phosphorylated kanamycin A has directly demonstrated regiospecific phosphoryl transfer to the 3'-hydroxyl of the 6-aminohexose ring of the antibiotic. Analysis of structure-activity relationships with a variety of aminoglycosides has revealed that the deoxystreptamine aminocyclitol ring plays a critical role in substrate binding. This information will form the basis for future design of inhibitors of APH(3')-IIIa.

Aminoglycosides↗
PubMed1994

D-penicillamine induced polymyositis causing complete heart block.

We describe a 63-year-old female patient with rheumatoid arthritis who developed complete heart block and features of polymyositis within a few weeks of starting treatment with D-penicillamine. We believe she is one of only three published patients in whom complete heart block accompanies penicillamine-induced polymyositis. The literature on penicillamine myositis is reviewed with special emphasis on cardiac problems. Patients taking D-penicillamine who develop features suggestive of polymyositis may develop insidious, but potentially life-threatening cardiac involvement and must be carefully monitored.

Arthritis, Rheumatoid↗