Zinc-thiolate intermediate in catalysis of methyl group transfer in Methanosarcina barkeri.

Methyl group transfer reactions are essential in methane-forming pathways in all methanogens. The involvement of zinc in catalysis of methyl group transfer was studied for the methyltransferase enzyme MT2-A important for methanogenesis in Methanosarcina barkeri growing on methylamines. Zinc was shown to be required for MT2-A activity and was tightly bound by the enzyme with an apparent stability constant of 10(13.7) at pH 7.2. Oxidation was a factor influencing activity and metal stoichiometry of purified MT2-A preparations. Methods were developed to produce inactive apo MT2-A and to restore full activity with stoichiometric reincorporation of Zn(2+). Reconstitution with Co(2+) yielded an enzyme with 16-fold higher specific activity. Cysteine thiolate coordination in Co(2+)-MT2-A was indicated by high absorptivity in the 300-400 nm charge transfer region, consistent with more than one thiolate ligand at the metal center. Approximate tetrahedral geometry was indicated by strong d-d transition absorbance centered at 622 nm. EXAFS analyses of Zn(2+)-MT2-A revealed 2S + 2N/O coordination with evidence for involvement of histidine. Interaction with the substrate CoM (2-mercaptoethanesulfonic acid) resulted in replacement of the second N/O group with S, indicating direct coordination of the CoM thiolate. UV-visible spectroscopy of Co(2+)-MT2-A in the presence of CoM also showed formation of an additional metal-thiolate bond. Binding of CoM over the range of pH 6.2-7.7 obeyed a model in which metal-thiolate formation occurs separately from H(+) release from the enzyme-substrate complex. Proton release to the solvent takes place from a group with apparent pK(a) of 6.4, and no evidence for metal-thiolate protonation was found. It was determined that substrate metal-thiolate bond formation occurs with a Delta G degrees ' of -6.7 kcal/mol and is a major thermodynamic driving force in the overall process of methyl group transfer.

The molecular basis for the natural resistance of the cytochrome bc1 complex from strobilurin-producing basidiomycetes to center Qp inhibitors.

Mitochondria from the strobilurin A producing basidiomycetes Strobilurus tenacellus and Mycena galopoda exhibit natural resistance to (E)-beta-methoxyacrylate inhibitors of the ubiquinol oxidation center(center Qp) of the cytochrome bc1 complex. Isolated cytochrome bc1 complex from S. tenacellus was found to be highly similar to that of Saccharomyces cerevisiae with respect to subunit composition, as well as spectral characteristics and midpoint potentials of the heme centers. To understand the molecular basis of natural resistance, we determined the exon/intron organization and deduced the sequences of cytochromes b from S. tenacellus, M. galopoda and a third basidiomycete, Mycena viridimarginata, which produces no strobilurin A. Comparative sequence analysis of two regions of cytochrome b known to contribute to the formation of center Qp suggested that the generally lower sensitivity of all three basidiomycetes was due to the replacement of a small amino acid residue in position 127 by isoleucine. For M. galopoda replacement of Gly143 by alanine and Gly153 by serine, for S. tenacellus replacement of a small residue in position 254 by glutamine and Asn261 by aspartate was found to be the likely causes for resistance to (E)-beta-methoxyacrylates. The latter exchange is also found in Schizosaccharomyces pombe, which we found also to be naturally resistant to (E)-beta-methoxyacrylates.

Human diseases with defects in oxidative phosphorylation. 1. Decreased amounts of assembled oxidative phosphorylation complexes in mitochondrial encephalomyopathies.

The amount of oxidative phosphorylation enzymes in mitochondrial encephalomyopathy patients has been studied by two-dimensional electrophoresis (blue native PAGE/Tricine-SDS-PAGE). Only 20 mg muscle was required to identify and analyse complexes I, III, IV, and V after Coomassie staining. In most cases reduced amounts of the involved complex(es) correlated well with decreased enzyme activities. The reliability of the method was reflected by the constant mutual ratio of the complexes found in all controls. Deviations from normal ratios were found to be more sensitive indicators for a defect than the absolute quantities, which varied considerably within the control group both in the enzymic and in the electrophoretic analysis. The effect of the mitochondrial tRNA(Leu(UUR)) mutation in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes on the amount of oxidative phosphorylation complexes was demonstrated for the first time directly on the protein level. In patients without known DNA mutations, specific defects of single complexes were identified. The new technique is a sensitive method for the identification of oxidative phosphorylation defects, complementary to enzymic measurements.

Core I protein of bovine ubiquinol-cytochrome-c reductase; an additional member of the mitochondrial-protein-processing family. Cloning of bovine core I and core II cDNAs and primary structure of the proteins.

Core I and core II proteins are the largest nuclear-encoded subunits of the mitochondrial ubiquinol-cytochrome-c reductase (bc1 complex) lacking redox prosthetic groups. cDNA clones of the two bovine core proteins have been isolated by the screening of lambda ZAP cDNA libraries either with an oligonucleotide probe based on the sequence of an internal peptide or with a polymerase-chain-reaction-amplified fragment. The core I precursor protein consists of 362 amino acids with a 34-amino-acid presequence typical for mitochondrial targeting signals. The mature protein migrates in SDS/polyacrylamide gels with an apparent molecular mass of 47 kDa, which does not correspond to the actual molecular mass of the protein of 35.8 kDa deduced from the cDNA sequence. The core II precursor protein is composed of 453 amino acids having a 14-amino-acid presequence as a targeting sequence. Comparison of the core I amino acid sequence with sequences of the newly discovered protein family [Schulte, U., Arretz, M., Schneider, H., Tropschug, M., Wachter E., Neupert, W. & Weiss, H. (1989) Nature 339, 147 - 149] comprising the processing enhancing protein (PEP), matrix processing peptidase (MPP), and core I and II proteins from Neurospora crassa and Saccharomyces cerevisiae, revealed a remarkable identity of 39% and a high similarity of 49% to N. crassa PEP, which in this fungus is identical to core I. Core II protein is only a distant relative of this protein family. Based on these sequence comparisons and data obtained by genomic Southern blots, we anticipate that the bovine core I subunit, like the N. crassa core I protein, is bifunctional, being responsible for the maintenance of electron transport and processing of proteins during their import into the mitochondrial matrix. The analysis of the primary structure of the two core proteins completes the set of primary structures of all subunits of bovine ubiquinol-cytochrome-c reductase.

Conservative amino acid substitution in the myelin proteolipid protein of jimpymsd mice.

The capacity for synthesizing and maintaining a compact myelin sheath is destroyed in a number of inborn errors of myelin metabolism. One class of hypomyelinating mutations, which displays an X-linked pattern of inheritance, is distinguished by marked disturbances in oligodendrocyte differentiation. We have defined the molecular defect in one such mutant that lacks mature oligodendrocytes, the X-linked jimpy myelin synthesis deficient (jpmsd) trait in mice. The structure of the gene encoding the most abundant myelin protein, proteolipid protein (PLP), was determined by mapping and partially sequencing genomic clones from jpmsd and wild-type mice. Jpmsd mice have a single base change in PLP, a C----T transition in exon 6 that would substitute a valine for alanine in both PLP and its alternatively spliced isoform, DM20. The mutation was confirmed by polymerase chain reaction-amplifying exon 6 from genomic DNA and then either sequencing the amplified DNA or directly probing exon 6 with oligonucleotides designed to detect a single base mismatch. The conservative amino acid replacement in PLP/DM20 of jpmsd mice results in a pleiotropic phenotype similar to that observed for the allelic mutation jimpy, in which a splicing defect has radically altered the PLP/DM20 protein. The accelerated turnover of oligodendrocytes in both mouse mutants suggests a function for PLP/DM20 in oligodendrocyte differentiation distinct from the role of these proteolipid proteins as structural components of the myelin sheath.

Mutation of the proteolipid protein gene PLP in a human X chromosome-linked myelin disorder.

Myelin is a highly specialized membrane unique to the nervous system that ensheaths axons to permit the rapid saltatory conduction of impulses. The elaboration of a compact myelin sheath is disrupted in a diverse spectrum of human disorders, many of which are of unknown etiology. The X chromosome-linked human disorder Pelizaeus-Merzbacher disease is a clinically and pathologically heterogeneous group of disorders that demonstrate a striking failure of oligodendrocyte differentiation. This disease appears pathologically and genetically to be similar to the disorder seen in the dysmyelinating mouse mutant jimpy, which has a point mutation in the gene encoding an abundant myelin protein, proteolipid protein (PLP). We report that the molecular defect in one Pelizaeus-Merzbacher family is likewise a point mutation in the PLP gene. A single T----C transition results in the substitution of a charged amino acid residue, arginine, for tryptophan in one of the four extremely hydrophobic domains of the PLP protein. The identification of a mutation in this Pelizaeus-Merzbacher family should facilitate the molecular classification and diagnosis of these X chromosome-linked human dysmyelinating disorders.

Pelizaeus-Merzbacher disease: an X-linked neurologic disorder of myelin metabolism with a novel mutation in the gene encoding proteolipid protein.

The nosology of the inborn errors of myelin metabolism has been stymied by the lack of molecular genetic analysis. Historically, Pelizaeus-Merzbacher disease has encompassed a host of neurologic disorders that present with a deficit of myelin, the membrane elaborated by glial cells that encircles and successively enwraps axons. We describe here a Pelizaeus-Merzbacher pedigree of the classical type, with X-linked inheritance, a typical clinical progression, and a pathologic loss of myelinating cells and myelin in the central nervous system. To discriminate variants of Pelizaeus-Merzbacher disease, a set of oligonucleotide primers was constructed to polymerase-chain-reaction (PCR) amplify and sequence the gene encoding proteolipid protein (PLP), a structural protein that comprises half of the protein of the myelin sheath. The PLP gene in one of two affected males and the carrier mother of this family exhibited a single base difference in the more than 2 kb of the PLP gene sequenced, a C----T transition that would create a serine substitution for proline at the carboxy end of the protein. Our results delineate the clinical features of Pelizaeus-Merzbacher disease, define the possible molecular pathology of this dysmyelinating disorder, and address the molecular classification of inborn errors of myelin metabolism. Patients with the classical form (type I) and the more severely affected, connatal variant of Pelizaeus-Merzbacher disease (type II) would be predicted to display mutation at the PLP locus. The other variants (types III-VI), which have sometimes been categorized as Pelizaeus-Merzbacher disease, may represent mutations in genes encoding other structural myelin proteins or proteins critical to myelination.

Transactivation of several genes of two native Serratia prophages after superinfection by phage kappa.

Serratia marcescens HY bacteria must be lysogenic with either prophage y or psi to make it possible for phage kappa to form plaques unless they carry a so-called ink mutation. Genes in y and psi termed any and anp were identified that after infection of ink+ cells are necessary for an effective propagation of these phages as well as of coinfecting kappa phage. When kappa infects y and/or psi-lysogenic cells it transactivates the respective prophage genes by means of two early genes termed tay and tap. It appears that on infection of nonlysogenic ink+ cells kappa damps its own development, provided the regulatory region of the responsible gene is undermethylated. After kappa infection duly to achieve the special methylation of this region seems to be the function of any and anp. There are some more genes in y and psi prophage under the control of tay and tap, concerning in both cases a Dam methylation (recognition sequence GATC) of kappa DNA, a recombination proneness under restricting conditions of kappa DNA not modified by the modification enzyme of HY, and the kappa plaque size. By hybridization studies a region of homology common to y and psi was demonstrated which from its size might comprise all the transactivated genes. The view is supported by genetic data indicating an affinity among the any and anp genes. Investigation of various any mutants were indicative of DNA inversions in this region of the y genome. Surprisingly some of the any mutants had become sensitive in their plaque forming ability to an inhibitory activity exerted by prophage psi. Mutants of psi unable to interfere but still able to lysogenize were isolated. A model is presented accounting for the formation of pleiotropic and nonpleiotropic mutations with Any phenotype and their reversion types. Possible functions of the y genes and their counterparts in psi are discussed.