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Titolo:
A THIOREDOXIN FUSION PROTEIN OF VANH, A D-LACTATE DEHYDROGENASE FROM ENTEROCOCCUS-FAECIUM - CLONING, EXPRESSION, PURIFICATION, KINETIC-ANALYSIS, AND CRYSTALLIZATION
Autore:
STOLL VS; MANOHAR AV; GILLON W; MACFARLANE ELA; HYNES RC; PAI EF;
Indirizzi:
UNIV TORONTO,DEPT BIOCHEM,1 KINGS COLL CIRCLE TORONTO ON M5S 1A8 CANADA UNIV TORONTO,DEPT BIOCHEM TORONTO ON M5S 1A8 CANADA UNIV TORONTO TORONTO ON M5G 2M9 CANADA UNIV TORONTO,MOL & MED GENET & PROT ENGN NETWORK CTR EXCELLENC TORONTO ON M5S 1A8 CANADA ONTARIO CANC INST,DIV MOL & STRUCT BIOL TORONTO ON M5G 2M9 CANADA
Titolo Testata:
Protein science
fascicolo: 5, volume: 7, anno: 1998,
pagine: 1147 - 1155
SICI:
0961-8368(1998)7:5<1147:ATFPOV>2.0.ZU;2-T
Fonte:
ISI
Lingua:
ENG
Soggetto:
DEPSIPEPTIDE PEPTIDOGLYCAN PRECURSORS; VANCOMYCIN RESISTANCE; GLYCOPEPTIDE RESISTANCE; SUBSTRATE-BINDING; CRYSTAL-STRUCTURE; BM4147; RESOLUTION; D-,D-DIPEPTIDASE; SPECIFICITY; TN1546;
Keywords:
ANTIBIOTIC RESISTANCE; D-LACTATE DEHYDROGENASE; ENTEROCOCCUS FAECIUM; ENZYME KINETICS; PROTEIN CRYSTALLIZATION; VANCOMYCIN;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
36
Recensione:
Indirizzi per estratti:
Citazione:
V.S. Stoll et al., "A THIOREDOXIN FUSION PROTEIN OF VANH, A D-LACTATE DEHYDROGENASE FROM ENTEROCOCCUS-FAECIUM - CLONING, EXPRESSION, PURIFICATION, KINETIC-ANALYSIS, AND CRYSTALLIZATION", Protein science, 7(5), 1998, pp. 1147-1155

Abstract

The gene encoding the vancomycin resistance protein VanH from Enterococcus faecium, a D-lactate dehydrogenase, has been cloned into a thioredoxin expression system (pTRxFus) and expressed as a fusion protein. The use of several other expression systems yielded only inclusion bodies from which no functional protein could be recovered. Experiments to remove the thioredoxin moiety by enterokinase cleavage at the engineered recognition site under a variety of conditions resulted in nonspecific proteolysis and inactivation of the protein. The intact fusion protein was, therefore, used for kinetic studies and crystallization trials. It has been purified to greater than 90% homogeneity by ammoniumsulfate precipitation followed by phenyl Sepharose chromatography. Based on k(cat)/K-M for pyruvate, it is 20% as active as native VanH. Michaelis constants for NADPH, NADH, and pyruvate, of similar to 3.5 mu M, 19.0 mu M, and 1.5 mM, respectively, were comparable to those reported for the native VanH (Bugg TDH et al., 1991, Biochemistry 30:10408-10415). Like native VanH, maximum activity of the fusion protein requires the presence of an anion (phosphate or acetate), however, in addition, a strongly reducing environment is needed for optimal efficacy. Competitive inhibition constants for ADP-ribose, NAD(+), and oxamate have also been determined. Crystallization by hanging drop vapor diffusion produced two different crystal forms, one hexagonal and the other tetragonal. Flash-frozen crystals of the tetragonal form diffracted to 3.0 Angstrom resolution at a synchrotron radiation source.

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Documento generato il 26/11/20 alle ore 19:19:34