Genetic and Biochemical Characterization of the Lipid-Protein Interactions of Pyruvate Oxidase
Grabau, Charlotte Louise
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https://hdl.handle.net/2142/71184
Description
Title
Genetic and Biochemical Characterization of the Lipid-Protein Interactions of Pyruvate Oxidase
Author(s)
Grabau, Charlotte Louise
Issue Date
1987
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Abstract
The pyruvate oxidase structural gene (poxB) of Escherichia coli was cloned into derivatives of plasmid pBR322. The gene was first cloned into a cosmid vector by selection for the tetracycline resistance determinant of a closely linked Tn 10 insertion since no direct selection was available. Subsequent subcloning resulted in localization of the gene to a 3.2-kilobase-pair DNA segment, which was shown to cause the overproduction of oxidase activity (by six- to eightfold) and encode a protein having the size and antigenic determinants of pyruvate oxidase.
The entire necleotide sequence of the poxB gene was determined by sequencing fragments of the gene cloned into a phage M13 vector. The gene is 1716 nucleotides in length and has an open reading frame which encodes a protein of Mr 62,018. This open reading frame was shown to encode pyruvate oxidase by alignment of the amino acid sequences deduced for the amino and carboxy termini and several internal segments of the mature protein with previously reported sequences obtained by amino acid sequence analysis. The deduced amino acid sequence of the oxidase was not unusually rich in hydrophobic sequences despite the peripheral membrane location and lipid binding properties of the protein. The deduced amino acid sequence shares 30% homology with the large subunit of the acetohydroxy acid synthase isozymes I, II, and III, encoded by the ilvB, ilvG, and ilvI genes of E. coli.
To differentiate between activation and lipid binding, I have constructed, using recombinant DNA techniques, a mutant gene that produces a truncated protein lacking the last 24 amino acids of the C-terminus of the oxidase and is thus closely analogous to the activated species produced in vitro by limited chymotrypsin cleavage. The truncated protein is fully active in vitro in the absence of lipid, and its activity is not further increased by addition of lipid activators. Moreover, the truncated enzyme fails to bind Triton X-114, a detergent that binds to and activates the wild type oxidase. Strains producing the truncated protein were devoid of pyruvate oxidase activity in vivo. This result indicates that binding to membrane lipids is specifically required for function of pyruvate oxidase in vivo; activation alone does not suffice.
To address what portion of this region (the last 24 amino acids) was involved in lipid binding, two additional mutants were constructed by similar mutagenesis techniques, producing proteins lacking the last nine or three amino acids from the end of the protein. Both truncated proteins were unable to interact with lipid, indicating that the last three amino acids play a role in the association of pyruvate oxidase with lipids.
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