臭味假单胞菌乙酰乙酰辅酶A硫解酶的提纯及性质研究

从臭味假单胞菌中提纯97倍的AcAcCoA硫解酶在聚丙烯酰胺凝胶电泳上是均一的一带。该酶分子量为170,000,每分子含有4个亚基,亚基分子量为42,000。该酶的等电点为pI6.7。它的N-末端为丙氨酸,N-末端是单一的。该酶催化反应的Km值为10.2μmol/L,最大反应速度为16.7μmol/min·mg。 臭味假单胞菌细胞粗提液透析后,经DEAE-纤维素(DE-52)柱色谱,从洗脱液中可同时得到四个酶的活力峰:乙酰乙酸琥珀酰辅酶A转移酶,AcAcCoA硫解酶,β-酮已二酸琥珀酰辅酶A转移酶和β-酮己二酸单酰辅酶A硫解酶。一般认为在细菌的芳径代谢中存在β-酮己二酸代谢途径,上述四个酶的活力峰同时存在说明除β-酮已二酸代谢途径外,还同时存在乙酰乙酸代谢途径。     

Phylogenetic analysis of the thiolase family. Implications for the evolutionary origin of peroxisomes

Summary The thiolase family is a widespread group of proteins present in prokaryotes and three cellular compartments of eukaryotes. This fact makes this family interesting in order to study the evolutionary process of eukaryotes. Using the sequence of peroxisomal thiolase from Saccharomyces cerevisiae recently obtained by us and the other known thiolase sequences, a phylogenetic analysis has been carried out. It shows that all these proteins derived from a primitive enzyme, present in the common ancestor of eubacteria and eukaryotes, which evolved into different specialized thiolases confined to various cell compartments. The evolutionary tree obtained is compatible with the endosymbiotic theory for the origin of peroxisomes. Offprint requests to: J.E. Pérez-Ortín     

Genome analysis of a hyper acetone‐butanol‐ethanol (ABE) producing Clostridium acetobutylicum BKM19

Previously the development of a hyper acetone‐butanol‐ethanol (ABE) producing Clostridium acetobutylicum BKM19 strain capable of producing 30.5% more total solvent by random mutagenesis of its parental strain PJC4BK, which is a buk mutant C. acetobutylicum ATCC 824 strain is reported. Here, BKM19 and PJC4BK strains are re‐sequenced by a high‐throughput sequencing technique to understand the mutations responsible for enhanced solvent production. In comparison with the C. acetobutylicum PJC4BK, 13 single nucleotide variants (SNVs), one deletion and one back mutation SNV are identified in the C. acetobutylicum BKM19 genome. Except for one SNV found in the megaplasmid, all mutations are found in the chromosome of BKM19. Among them, a mutation in the thlA gene encoding thiolase is further studied with respect to enzyme activity and butanol production. The mutant thiolase (thlA^V5A) is showed a 32% higher activity than that of the wild‐type thiolase (thlA^WT). In batch fermentation, butanol production is increased by 26% and 23% when the thlA^V5A gene is overexpressed in the wild‐type C. acetobutylicum ATCC 824 and in its derivative, the thlA‐knockdown TKW‐A strain, respectively. Based on structural analysis, the mutation in thiolase does not have a direct effect on the regulatory determinant region (RDR). However, the mutation at the 5^th residue seems to influence the stability of the RDR, and thus, increases the enzymatic activity and enhances solvent production in the BKM19 strain.     

Up-regulation of the peroxisomal β-oxidation system occurs in butyrate-grown Candida tropicalis following disruption of the gene encoding peroxisomal 3-ketoacyl-CoA thiolase

In the yeast Candida tropicalis, two thiolase isozymes, peroxisomal acetoacetyl-CoA thiolase and peroxisomal 3-ketoacyl-CoA thiolase, participate in the peroxisomal fatty acid β-oxidation system. Their individual contributions have been demonstrated in cells grown on butyrate, with C. tropicalis able to grow in the absence of either one. In the present study, a lack of peroxisomal 3-ketoacyl-CoA thiolase protein resulted in increased expression (up-regulation) of acetoacetyl-CoA thiolase and other peroxisomal proteins, whereas a lack of peroxisomal acetoacetyl-CoA thiolase produced no corresponding effect. Overexpression of the acetoacetyl-CoA thiolase gene did not suppress the up-regulation or the growth retardation on butyrate in cells without peroxisomal 3-ketoacyl-CoA thiolase, even though large amounts of the overexpressed acetoacetyl-CoA thiolase were detected in most of the peroxisomes of butyrate-grown cells. These results provide important evidence of the greater contribution of 3-ketoacyl-CoA thiolase to the peroxisomal β-oxidation system than acetoacetyl-CoA thiolase in C. tropicalis and a novel insight into the regulation of the peroxisomal β-oxidation system.     

The central metabolic pathway from acetyl-CoA to butyryl-CoA in Clostridium acetobutylicum

Abstract: The pathway from acetyl-CoA to butyryl-CoA serves as a major carbon metabolism channel in Clostridium acetobutylicum and other butyrate-forming clostridia, and the steps are similar to those involved in fatty acid metabolism. Recent findings are discussed, reviewing the isolation and characterization of the enzymes of the pathway, and the analyses of metabolic intermediate levels and possible points of regulation of enzyme activity by CoA compounds. DNA analyses have identified the genes for two thiolase proteins, and an apparent operon encoding five proteins involved in the conversion of acetoacetyl-CoA to butyryl-CoA. These five proteins are β-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase and the α and β subunits of an electron transfer flavoprotein.     

Phylogenetic Analysis of Eukaryotic Thiolases Suggests Multiple Proteobacterial Origins

Eukaryotic thiolases are essential enzymes located in three different compartments (peroxisome, mitochondrion, and cytosol) that can display catabolic or anabolic functions. They are responsible for the thiolytic cleavage of oxidized acyl-CoA (thiolase I; EC 2.3.1.16) and the synthesis or degradation of acetoacetyl-CoA (thiolase II; EC 2.3.1.9). Phylogenetic analysis of eukaryotic thiolase sequences showed that they form six distinct clusters, one of them highly divergent, which are in good correlation with their class and subcellular location. When analyzed together with a representative sample of prokaryotic thiolases, all eukaryotic thiolase groups emerged close to proteobacterial sequences. Metazoan cytosolic thiolase II was related to α-proteobacterial sequences, suggesting a mitochondrial origin. Unexpectedly, cytosolic thiolases from green plants and fungi as well as at least one member of all eukaryotic peroxisomal and mitochondrial thiolases had δ-proteobacteria as closest relatives. Our analysis suggests that these eukaryotic peroxisomal and mitochondrial thiolases may have been acquired from δ-proteobacteria prior to the ancestor of all known eukaryotes.     

Purification and characterization of OleA from Xanthomonas campestris and demonstration of a non-decarboxylative Claisen condensation reaction

OleA catalyzes the condensation of fatty acyl groups in the first step of bacterial long-chain olefin biosynthesis, but the mechanism of the condensation reaction is controversial. In this study, OleA from Xanthomonas campestris was expressed in Escherichia coli and purified to homogeneity. The purified protein was shown to be active with fatty acyl-CoA substrates that ranged from C(8) to C(16) in length. With limiting myristoyl-CoA (C(14)), 1 mol of the free coenzyme A was released/mol of myristoyl-CoA consumed. Using [(14)C]myristoyl-CoA, the other products were identified as myristic acid, 2-myristoylmyristic acid, and 14-heptacosanone. 2-Myristoylmyristic acid was indicated to be the physiologically relevant product of OleA in several ways. First, 2-myristoylmyristic acid was the major condensed product in short incubations, but over time, it decreased with the concomitant increase of 14-heptacosanone. Second, synthetic 2-myristoylmyristic acid showed similar decarboxylation kinetics in the absence of OleA. Third, 2-myristoylmyristic acid was shown to be reactive with purified OleC and OleD to generate the olefin 14-heptacosene, a product seen in previous in vivo studies. The decarboxylation product, 14-heptacosanone, did not react with OleC and OleD to produce any demonstrable product. Substantial hydrolysis of fatty acyl-CoA substrates to the corresponding fatty acids was observed, but it is currently unclear if this occurs in vivo. In total, these data are consistent with OleA catalyzing a non-decarboxylative Claisen condensation reaction in the first step of the olefin biosynthetic pathway previously found to be present in at least 70 different bacterial strains.     

Crystal structure and biochemical characterization of PhaA from Ralstonia eutropha, a polyhydroxyalkanoate-producing bacterium

PhaA from Ralstonia eutropha (RePhaA) is the first enzyme in the polyhydroxyalbutyrate (PHB) biosynthetic pathway and catalyzes the condensation of two molecules of acetyl-CoA to acetoacetyl-CoA. To investigate the molecular mechanism underlying PHB biosynthesis, we determined the crystal structures of the RePhaA protein in apo- and CoA-bound forms. The RePhaA structure adopts the type II biosynthetic thiolase fold forming a tetramer by means of dimerization of two dimers. The crystal structure of RePhaA in complex with CoA revealed that the enzyme contained a unique Phe219 residue, resulting that the ADP moiety binds in somewhat different position compared with that bound in other thiolase enzymes. Our study provides structural insight into the substrate specificity of RePhaA. Results indicate the presence of a small pocket near the Cys88 covalent catalytic residue leading to the possibility of the enzyme to accommodate acetyl-CoA as a sole substrate instead of larger acyl-CoA molecules such as propionyl-CoA. Furthermore, the roles of key residues involved in substrate binding and enzyme catalysis were confirmed by site-directed mutagenesis.     

Peroxisomes in mouse and human lung: their involvement in pulmonary lipid metabolism

Only sparse information is available from the literature on the peroxisomal compartment and its enzyme composition in mouse and human lungs. Therefore, in the present investigation we have characterized peroxisomes in different cell types of adult mouse (C57BL/6J) and human lungs in a comprehensive study using a variety of light-, fluorescence- and electron microscopic as well as biochemical techniques and by the use of various peroxisomal marker proteins (Pex13p, Pex14p, ABCD3, beta-oxidation enzymes and catalase). In contrast to previous reports, we have found that peroxisomes are present in all cell types in human and mouse lungs. However, they differ significantly and in a cell-type-specific manner in their structure, numerical abundance and enzyme composition. Whereas catalase showed significant differences between distinct cell types, Pex14p proved to be the marker of choice for labeling all lung peroxisomes. In alveolar type II cells and alveolar macrophages peroxisomes contained significant amounts of the lipid transporter ABCD3 and beta-oxidation enzymes, suggesting their involvement in the modification and recycling of surfactant lipids and in the control of lipid mediators and ligands for nuclear receptors of the PPAR family. Possible connections between ROS and lipid metabolism of lung peroxisomes are discussed.