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Titolo:
Substitution, insertion, deletion, suppression, and altered substrate specificity in functional protocatechuate 3,4-dioxygenases
Autore:
DArgenio, DA; Vetting, MW; Ohlendorf, DH; Ornston, LN;
Indirizzi:
Yale Univ, Dept Mol Cellular & Dev Biol, New Haven, CT 06520 USA Yale Univ New Haven CT USA 06520 ular & Dev Biol, New Haven, CT 06520 USA Univ Minnesota, Sch Med, Ctr Met Biocatalysis, Minneapolis, MN 55455 USA Univ Minnesota Minneapolis MN USA 55455 alysis, Minneapolis, MN 55455 USA Univ Minnesota, Sch Med, Dept Biochem Mol Biol & Biophys, Minneapolis, MN 55455 USA Univ Minnesota Minneapolis MN USA 55455 iophys, Minneapolis, MN 55455 USA
Titolo Testata:
JOURNAL OF BACTERIOLOGY
fascicolo: 20, volume: 181, anno: 1999,
pagine: 6478 - 6487
SICI:
0021-9193(199910)181:20<6478:SIDSAA>2.0.ZU;2-C
Fonte:
ISI
Lingua:
ENG
Soggetto:
P-HYDROXYBENZOATE HYDROXYLASE; SP. STRAIN ADP1; ACINETOBACTER-CALCOACETICUS; PROTEIN STABILITY; GENETIC-ANALYSIS; EVOLUTIONARY DIVERGENCE; PSEUDOMONAS-PUTIDA; NATURAL TRANSFORMATION; BENZOATE DEGRADATION; ESCHERICHIA-COLI;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
60
Recensione:
Indirizzi per estratti:
Indirizzo: Ornston, LN Yale Univ, Dept Mol Cellular & Dev Biol, POB 208103, New Haven, CT 06520 USA Yale Univ POB 208103 New Haven CT USA 06520 aven, CT 06520 USA
Citazione:
D.A. D'Argenio et al., "Substitution, insertion, deletion, suppression, and altered substrate specificity in functional protocatechuate 3,4-dioxygenases", J BACT, 181(20), 1999, pp. 6478-6487

Abstract

Protocatechuate 3,4-dioxygenase is a member of a family of bacterial enzymes that cleave the aromatic rings of their substrates between two adjacent hydroxyl groups, a key reaction in microbial metabolism of varied environmental chemicals. In an appropriate genetic background, it is possible to select for Acinetobacter strains containing spontaneous mutations blocking expression of pcaH or -G, genes encoding the or and beta subunits of protocatechuate 3,4-dioxygenase. The crystal structure of the Acinetobacter oxygenase has been determined, and this knowledge affords us the opportunity to understand how mutations alter function in the enzyme. An earlier investigation had shown that a large fraction of spontaneous mutations inactivating Acinetobacter protocatechuate oxygenase are either insertions or large deletions. Therefore, the prior procedure of mutant selection was modified to isolate Acinetobacter strains in which mutations within pcaH or -G cause a heat-sensitive phenotype, These mutations affected residues distributed throughout the linear amino acid sequences of PcaH and PcaG and impaired the dioxygenase to various degrees. Four of 16 mutants had insertions or deletions in the enzyme ranging in size from 1 to 10 amino acid residues, highlightingareas of the protein where large structural changes can be tolerated. To further understand how protein structure influences function, we isolated strains in which the phenotypes of three different deletion mutations in pcaHor -G were suppressed either by a spontaneous mutation or by a PCR-generated random mutation introduced into the Acinetobacter chromosome by natural transformation. The latter procedure was also used to identify a single amino acid substitution in PcaG that conferred activity towards catechol sufficient for growth with benzoate in a strain in which catechol 1,2-dioxygenase was inactivated.

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Documento generato il 28/03/20 alle ore 21:58:47