ABSTRACT
Mia40 participates in oxidative protein folding within the mitochondrial intermembrane space (IMS) by mediating the transfer of reducing equivalents from client proteins to FAD-linked oxidoreductases of the Erv1 family (lfALR in mammals). Here we investigate the specificity of the human Mia40/lfALR system towards non-cognate unfolded protein substrates to assess whether the efficient introduction of disulfides requires a particular amino acid sequence context or the presence of an IMS targeting signal. Reduced pancreatic ribonuclease A (rRNase), avian lysozyme, and riboflavin binding protein are all competent substrates of the Mia40/lfALR system, although they lack those sequence features previously thought to direct disulfide bond formation in cognate IMS substrates. The oxidation of rRNase by Mia40 does not limit overall turnover of unfolded substrate by the Mia40/lfALR system. Mia40 is an ineffective protein disulfide isomerase when its ability to restore enzymatic activity from scrambled RNase is compared to that of protein disulfide isomerase. Mia40's ability to bind amphipathic peptides is evident by avid binding to the isolated B-chain during the insulin reductase assay. In aggregate these data suggest that the Mia40/lfALR system has a broad sequence specificity and that potential substrates may be protected from adventitious oxidation by kinetic sequestration within the mitochondrial IMS.
Subject(s)
Cytochrome Reductases/chemistry , Cytochrome Reductases/ultrastructure , Isomerases/chemistry , Isomerases/ultrastructure , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/ultrastructure , Amino Acid Sequence , Binding Sites , Computer Simulation , Enzyme Activation , Humans , Mitochondrial Precursor Protein Import Complex Proteins , Models, Chemical , Models, Molecular , Molecular Sequence Data , Oxidants/chemistry , Oxidation-Reduction , Oxidoreductases Acting on Sulfur Group Donors , Protein Binding , Protein Conformation , Protein Folding , Structure-Activity RelationshipABSTRACT
Methylibium petroleiphilum strain PM1 uses various petroleum products including the fuel additive methyl tert-butyl ether and straight chain and aromatic hydrocarbons as sole carbon and energy sources. It has two operons, dmpI and dmpII, that code for the enzymes in a pair of parallel meta-fission pathways. In order to understand the roles of the pathways, the 4-oxalocrotonate tautomerase (4-OT) isozyme from each pathway was characterized. Tautomerase I and tautomerase II have the lowest pairwise sequence identity (35%) among the isozyme pairs in the parallel pathways, and could offer insight into substrate preferences and pathway functions. The kinetic parameters of tautomerase I and tautomerase II were determined using 2-hydroxymuconate and 5-(methyl)-2-hydroxymuconate. Both tautomerase I and tautomerase II process the substrates, but with different efficiencies. Crystal structures were determined for both tautomerase I and tautomerase II, at 1.57 and 1.64Å resolution, respectively. The backbones of tautomerase I and tautomerase II are highly similar, but have distinct active site environments. The results, in combination with those for other structurally and kinetically characterized 4-OT isozymes, suggest that tautomerase I catalyzes the tautomerization of both 2-hydroxymuconate and alkyl derivatives, whereas tautomerase II might specialize in other aromatic hydrocarbon metabolites.
Subject(s)
Betaproteobacteria/classification , Betaproteobacteria/enzymology , Isomerases/chemistry , Isomerases/ultrastructure , Amino Acid Sequence , Enzyme Activation , Enzyme Stability , Kinetics , Molecular Sequence Data , Protein Conformation , Species Specificity , Substrate SpecificityABSTRACT
The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine. It is unprecedented in nature as it uses the cofactor S-adenosylmethionine as the donor of a ribosyl group. We have determined the crystal structure of Bacillus subtilis QueA at a resolution of 2.9A. The structure reveals two domains representing a 6-stranded beta-barrel and an alpha beta alpha-sandwich, respectively. All amino acid residues invariant in the QueA enzymes of known sequence cluster at the interface of the two domains indicating the localization of the substrate binding region and active center. Comparison of the B. subtilis QueA structure with the structure of QueA from Thermotoga maritima suggests a high domain flexibility of this enzyme.
Subject(s)
Bacillus subtilis/enzymology , Isomerases/chemistry , Isomerases/ultrastructure , Models, Chemical , Models, Molecular , Amino Acid Sequence , Binding Sites , Computer Simulation , Crystallography , Enzyme Activation , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Substrate SpecificityABSTRACT
The catalytic disulfide bond Cys30-Cys33 of the disulfide oxidoreductase DsbA from Escherichia coli is located at the amino terminus of an alpha-helix, which has a kink caused by insertion of a tripeptide (residues 38-40). The oxidative force of DsbA (E'O = -125 mV) mainly results from the low pKa of 3.4 of its Cys30 thiol. To investigate the role of the kink and the electrostatic contribution of Glu37 and Glu38 to the redox properties of DsbA, we have characterized a series of DsbA variants (delta38-40, delta38-40/H41P, E37Q, E38Q, and E37Q/E38Q). In contrast to theoretical predictions, the redox potentials of the variants are almost unchanged, and the pKa values of Cys30 do not differ by more than 0.5 units from that of DsbA wild type. All variants show the same in vivo activity and dependence of redox potential on ionic strength as the wild type. The mutations have no influence on the polypeptide specificity of the protein, which is independent of the isoelectric point of the polypeptide substrate and most pronounced at acidic pH. We conclude that neither the kink in the active-site helix nor Glu37 and Glu38 are critical for the physical properties of DsbA.
Subject(s)
Isomerases/chemistry , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/ultrastructure , Binding Sites , Disulfides/chemistry , Escherichia coli/enzymology , Glutamic Acid/chemistry , Glutamine/chemistry , Hydrogen-Ion Concentration , Ions , Isomerases/ultrastructure , Models, Molecular , Molecular Sequence Data , Osmolar Concentration , Point Mutation , Protein Denaturation , Protein Disulfide-Isomerases , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Deletion , Structure-Activity Relationship , Substrate Specificity , ThermodynamicsABSTRACT
The pJP4-encoded chloromuconate cycloisomerase, an enzyme of the 2,4-dichlorophenoxy-acetate degradation pathway, was purified from cell-free extracts of Alcaligenes eutrophus JMP134 with a revised procedure. Tetragonal bipyramidal crystals were grown and characterized with respect to their X-ray diffraction properties. They were assigned to the space group I4, with cell dimensions of a = b = 111.9 A, c = 148.5 A. The crystals scattered to approximately 3 A resolution.
Subject(s)
Alcaligenes/enzymology , Intramolecular Lyases , Isomerases/ultrastructure , Bacterial Proteins/isolation & purification , Bacterial Proteins/ultrastructure , Crystallography , Isomerases/isolation & purification , X-Ray DiffractionABSTRACT
By exhaustive structural comparisons, we have found that about one-third of the alpha-helix-turn-beta-strand polypeptides in alpha-beta barrel domains share a common structural motif. The chief characteristics of this motif are that first, the geometry of the turn between the alpha-helix and the beta-strand is somewhat constrained, and second, the beta-strand contains a hydrophobic patch that fits into a hydrophobic pocket on the alpha-helix. The geometry of the turn does not seem to be a major determinant of the alpha-beta unit, because the turns vary in length from four to six residues. However, the motif does not occur when there are few constraints on the geometry of the turn-for instance, when the turns between the alpha-helix and the beta-strands are very long. It also occurs much less frequently in flat-sheet alpha-beta proteins, where the topology is much less regular and the amount of twist on the sheet varies considerably more than in the barrel proteins. The motif may be one of the basic building blocks from which alpha-beta barrels are constructed.
