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Title
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Substitution, insertion, deletion, suppression, and altered substrate specificity in functional protocatechuate 3,4-dioxygenases.
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Authors
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D.A.D'Argenio,
M.W.Vetting,
D.H.Ohlendorf,
L.N.Ornston.
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Ref.
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J Bacteriol, 1999,
181,
6478-6487.
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PubMed id
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Abstract
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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 alpha 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, 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.
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