Substitution, insertion, deletion, suppression, and altered substrate specificity in functional protocatechuate 3,4-dioxygenases

David A. D'Argenio, Mathew W. Vetting, Douglas H. Ohlendorf, L. Nicholas Ornston

Research output: Contribution to journalArticle

28 Citations (Scopus)

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 α and β 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, highlighting areas 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 pcaH or -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.

Original languageEnglish (US)
Pages (from-to)6478-6487
Number of pages10
JournalJournal of Bacteriology
Volume181
Issue number20
StatePublished - Oct 1999
Externally publishedYes

Fingerprint

Protocatechuate-3,4-Dioxygenase
Substrate Specificity
Acinetobacter
Mutation
Oxygenases
Catechol 1,2-Dioxygenase
Enzymes
Phenotype
Dioxygenases
Sequence Deletion
Benzoates
Amino Acid Substitution
Hydroxyl Radical
Amino Acid Sequence
Proteins
Hot Temperature
Chromosomes
Amino Acids
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Immunology

Cite this

D'Argenio, D. A., Vetting, M. W., Ohlendorf, D. H., & Ornston, L. N. (1999). Substitution, insertion, deletion, suppression, and altered substrate specificity in functional protocatechuate 3,4-dioxygenases. Journal of Bacteriology, 181(20), 6478-6487.

Substitution, insertion, deletion, suppression, and altered substrate specificity in functional protocatechuate 3,4-dioxygenases. / D'Argenio, David A.; Vetting, Mathew W.; Ohlendorf, Douglas H.; Ornston, L. Nicholas.

In: Journal of Bacteriology, Vol. 181, No. 20, 10.1999, p. 6478-6487.

Research output: Contribution to journalArticle

D'Argenio, David A. ; Vetting, Mathew W. ; Ohlendorf, Douglas H. ; Ornston, L. Nicholas. / Substitution, insertion, deletion, suppression, and altered substrate specificity in functional protocatechuate 3,4-dioxygenases. In: Journal of Bacteriology. 1999 ; Vol. 181, No. 20. pp. 6478-6487.
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