Cloning, overexpression, and mutagenesis of the gene for homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum.
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.
Wang
Y Z
YZ
Department of Biochemistry, Medical School, University of Minnesota, Minneapolis 55455, USA.
Lipscomb
J D
JD
D015102:3,4-Dihydroxyphenylacetic Acid / Q000378:metabolism; D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D001673:Biodegradation, Environmental; D001951:Brevibacterium / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D003360:Cosmids / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D049308:Dioxygenases; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016368:Frameshift Mutation; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D005798:Genes, Bacterial; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010105:Oxygenases / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D015102:3,4-Dihydroxyphenylacetic Acid; D008345:Manganese; D007501:Iron; D010105:Oxygenases; D049308:Dioxygenases; C020055:3,4-dihydroxyphenylacetate 2,3-dioxygenase