On the lack of coordination between protein folding and flavin insertion in Escherichia coli for flavocytochrome b2 mutant forms Y254L and D282N

Muriel Gondry, K H Diêp Lê, Florence Lederer, Forbes D C Manson, Graeme A Reid, Stephen K Chapman, F Scott Mathews

    Research output: Contribution to journalArticle

    Abstract

    Wild-type flavocytochrome b(2) (L-lactate dehydrogenase) from Saccharomyces cerevisiae, as well as a number of its point mutants, can be expressed to a reasonable lever as recombinant proteins in Escherichia coli (20-25 mg per liter culture) with a full complement of prosthetic groups. At the same expression level, active-site mutants Y254L and D282N, on the other hand, were obtained with an FMN/heme ratio significantly less than unity, which could not be raised by addition of free FMN. Evidence is provided that the flavin deficit is due to incomplete prosthetic group incorporation during biosynthesis. Flavin-free and holo-forms for both mutants could be separated on a Blue-Trisacryl M column. The far-UV CD spectra of the two forms of each mutant protein were very similar to one another and to that of the wild-type enzyme, suggesting the existence of only local conformational differences between the active holo-enzymes and the nonreconstitutable flavin-free forms. Selective proteolysis with chymotrypsin attacked the same bond for the two mutant holo-enzymes as in the wild-type one, in the protease-sensitive loop. In contrast, for the flavin-free forms of both mutants, cleavage occurred at more than a single bond. Identification of the cleaved bonds suggested that the structural differences between the mutant flavin-free and holo-forms are located mostly at the C-terminal end of the barrel, which carries the prosthetic group and the active site. Altogether, these findings suggest that the two mutations induce an alteration of the protein-folding process during biosynthesis in E. coli; as a result, the synchrony between folding and flavin insertion is lost. Finally, a preliminary kinetic characterization of the mutant hole-forms showed the K-m value for lactate to be little affected; k(cat) values fell by a factor of about 70 for the D282N mutant and of more than 500 for the Y254L mutant, compared to the wild-type enzyme.

    Original languageEnglish
    Pages (from-to)925-935
    Number of pages11
    JournalProtein Science
    Volume4
    Issue number5
    DOIs
    Publication statusPublished - May 1995

    Keywords

    • RIBULOSE BISPHOSPHATE CARBOXYLASE
    • SELECTIVE PROTEOLYSIS
    • LACTATE DEHYDROGENATION
    • PROTEIN FOLDING
    • CYTOCHROME B2
    • RECOMBINANT PROTEIN
    • ACTIVE-SITE
    • CRYSTAL-STRUCTURE
    • MONONUCLEOTIDE-BINDING
    • SITE-DIRECTED MUTANTS
    • FLAVOENZYMES
    • HEAT-SHOCK PROTEINS
    • ACYL-COA DEHYDROGENASE
    • GLYCOLATE OXIDASE
    • BAKERS-YEAST
    • EXPRESSION
    • APOENZYME

    Cite this

    Gondry, M., Diêp Lê, K. H., Lederer, F., Manson, F. D. C., Reid, G. A., Chapman, S. K., & Mathews, F. S. (1995). On the lack of coordination between protein folding and flavin insertion in Escherichia coli for flavocytochrome b2 mutant forms Y254L and D282N. Protein Science, 4(5), 925-935. https://doi.org/10.1002/pro.5560040512