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Journal of Bacteriology, October 1999, p. 6478-6487, Vol. 181, No. 20
Department of Molecular, Cellular and
Developmental Biology, Yale University, New Haven, Connecticut
06520-8103,1 and Center for Metals in
Biocatalysis and Department of Biochemistry, Molecular Biology and
Biophysics, University of Minnesota Medical School, Minneapolis,
Minnesota 55455-03472
Received 14 May 1999/Accepted 21 July 1999
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
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Substitution, Insertion, Deletion, Suppression,
and Altered Substrate Specificity in Functional
Protocatechuate 3,4-Dioxygenases
and 
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.
*
Corresponding author. Mailing address: Department of
Molecular, Cellular and Developmental Biology, Yale University, P.O. Box 208103, New Haven, CT 06520-8103. Phone: (203) 432-3498. Fax: (203)
432-3497. E-mail: nicholas.ornston{at}yale.edu.
Publication 21 from the Biological Transformation Center in the
Yale Biospherics Institute.
Present address: Department of Genetics, University of Washington,
Seattle, WA 98195-7360.
Journal of Bacteriology, October 1999, p. 6478-6487, Vol. 181, No. 20
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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