title: Heterologous expression of enzymopenic methemoglobinemia variants using a novel NADH:cytochrome c reductase fusion protein.
abstract: Hereditary enzymopenic methemoglobinemia is a rare disease that predominantly results from defects in either the erythrocytic (type I) or microsomal (type II) forms of the enzyme NADH:cytochrome b5 reductase (EC 1.6.2.2). All 25 currently identified type I and type II methemoglobinemia mutants have been expressed in Escherichia coli using a novel six histidine-tagged rat cytochrome b5/cytochrome b5 reductase fusion protein designated NADH:cytochrome c reductase (H6NCR). All 25 H6NCR variants were isolated and demonstrated to result in two groups of expression products. The first group of 16 mutants, which included the majority of the type I mutants, included K116Q, P131L, L139P, T183S, M193V, S194P, P211L, L215P, A245T, A245V, C270Y, E279K, V305R, V319M, M340-, and F365-, and yielded full-length fusion proteins that retained variable levels of NADH:cytochrome c reductase (NADH:CR) activity, ranging from approximately 2% (M340-) to 92% (K116Q) of that of the wild-type fusion protein. In contrast, the remaining nine mutants that represented the majority of the type II variants, comprised a second group that included Y109*, R124Q, Q143*, R150*, P162H, V172M, R226*, C270R, and R285*, and resulted in truncated H6NCR variants that retained the amino-terminal cytochrome b5 domain but were devoid of NADH:CR activity due to the absence of the cytochrome b5 reductase flavin domain. Kinetic analyses of the first group of full-length mutant fusion proteins indicated that values for both kcat and Km(NADH) were decreased and increased, respectively, indicating that the various mutations affected both substrate affinity and/or turnover. However, for the second group, the truncated products were the result of incomplete production of the carboxyl-terminal flavin-containing domain or instability of the expression products due to improper folding and/or lack of flavin incorporation.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, FL 33612, USA.
authors.forename: C Ainsley
authors.initials: CA
authors.lastname: Davis
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Barber
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008708:Methemoglobinemia / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D009245:NADH Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
chemical_list: D011993:Recombinant Fusion Proteins; D009245:NADH Dehydrogenase