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Comparative Study
. 1997 Jun;10(1):1-9.
doi: 10.1006/prep.1996.0703.

Cloning, overexpression, and mutagenesis of the gene for homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum

Y Z Wang  1 , J D Lipscomb
Affiliations
Comparative Study

Cloning, overexpression, and mutagenesis of the gene for homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum

Y Z Wang et al. Protein Expr Purif. 1997 Jun.

Abstract

Homoprotocatechuate (hpca, 3,4-dihydroxyphenylacetate) is a central intermediate for the bacterial degradation of aromatic compounds. Homoprotocatechuate 2,3-dioxygenase (HPCD) catalyzes the key ring cleavage step in the metabolism of hpca by the Gram (+) bacterium Brevibacterium fuscum to yield alpha-hydroxy-delta-carboxymethyl cis-muconic semialdehyde. A genomic DNA library of B. fuscum was constructed in Escherichia coli using a cosmid vector and screened by spraying the cells with hpca. One clone was found to contain the gene for HPCD based on its ability to convert hpca into the yellow-colored product. This cosmid clone was further subcloned and the gene for HPCD was localized and sequenced. The open reading frame codes for a protein with 365 amino acids and M(r) = 41,699, in accord with the characteristics of the previously purified wild-type enzyme. The gene for HPCD was overexpressed in E. coli to approximately 30% of the total soluble protein, and purification of the recombinant enzyme to apparent homogeneity was achieved by a two-step procedure. Iron was the only abundant metal found in the purified recombinant enzyme, and the specific activity per iron was comparable to that observed for the wild-type enzyme. The deduced amino acid sequence of HPCD has a very high level of homology (78.6% identity in the 337-aa overlap) to the manganese-dependent homoprotocatechuate 2,3-dioxygenase (MndD) from Arthrobacter globiformis CM-2. The basis for the difference in metal selection by HPCD and MndD was investigated by mutagenesis of a 50-base-pair region of the HPCD gene containing three frame shifts relative to the MndD gene. The purified triple mutant of HPCD did not exhibit a significant change in the metal content; therefore, other factors must contribute to the selection of the active site metal.

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