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

Protein Expression and Purification
Y Z Wang, John D Lipscomb

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 ded...Continue Reading

References

Dec 1, 1977·Proceedings of the National Academy of Sciences of the United States of America·F SangerA R Coulson
Jan 1, 1990·Methods in Enzymology·J W WhittakerJ D Lipscomb
Dec 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·S Munro, T Maniatis
Mar 8, 1996·The Journal of Biological Chemistry·M A Miller, J D Lipscomb
Dec 9, 1964·Biochimica Et Biophysica Acta·K ADACHIH KITA

❮ Previous
Next ❯

Citations

Dec 15, 2010·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Andrew J FieldingLawrence Que
Feb 1, 2011·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Erik R FarquharLawrence Que
May 22, 2008·Proceedings of the National Academy of Sciences of the United States of America·Joseph P EmersonLawrence Que
Jul 28, 2010·Proceedings of the National Academy of Sciences of the United States of America·R TeufelG Fuchs
Sep 15, 2010·Proceedings of the National Academy of Sciences of the United States of America·Michael M MbughuniJohn D Lipscomb
Mar 19, 2004·Journal of Bacteriology·Matthew W VettingDouglas H Ohlendorf
Jul 5, 2013·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Joshua A HaydenMichael P Hendrich
May 28, 1998·The Journal of Biological Chemistry·C Su, E H Oliw
Jul 20, 2006·Critical Reviews in Biochemistry and Molecular Biology·Frédéric H VaillancourtLindsay D Eltis
Aug 24, 2012·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Kate L HendersonJoseph P Emerson
Jun 14, 2021·Pesticide Biochemistry and Physiology·Leily DaneshianMaksymilian Chruszcz

❮ Previous
Next ❯

Related Concepts

Related Feeds

Bioremediation (ASM)

Bioremediation is the treatment and removal of harmful pollutants or contaminants through the use of microorganisms. Discover the latest research here.