RESUMO
Taxoid 10ß-O-acetyl transferase (DBAT) is a key enzyme in the biosynthesis of the famous anticancer drug paclitaxel, which catalyses the formation of baccatin III from 10-deacetylbaccatin III (10-DAB). However, the activity essential residues of the enzyme are still unknown, and the acylation mechanism from its natural substrate 10-deacetylbaccatin III and acetyl CoA to baccatin III remains unclear. In this study, the homology modelling, molecular docking, site-directed mutagenesis, and kinetic parameter determination of the enzyme were carried out. The results showed that the enzyme mutant DBATH162A resulted in complete loss of enzymatic activity, suggesting that the residue histidine at 162 was essential to DBAT activity. Residues D166 and R363 which were located in the pocket of the enzyme by homology modelling and molecular docking were also important for DBAT activity through the site-directed mutations. Furthermore, four amino acid residues including S31 and D34 from motif SXXD, D372 and G376 from motif DFGWG also played important roles on acylation. This was the first report of the elucidation of the activity essential residues of DBAT, making it possible for the further structural-based re-design of the enzyme for efficient biotransformation of baccatin III and paclitaxel.
Assuntos
Acetilcoenzima A/química , Aldeído-Cetona Transferases/química , Alcaloides/síntese química , Simulação de Acoplamento Molecular , Proteínas de Plantas/química , Taxoides/síntese química , Taxus/enzimologia , Aldeído-Cetona Transferases/genética , Alcaloides/química , Substituição de Aminoácidos , Mutação de Sentido Incorreto , Paclitaxel/síntese química , Paclitaxel/química , Proteínas de Plantas/genética , Taxoides/química , Taxus/genéticaRESUMO
Pyruvylation onto the terminus of oligosaccharide, widely seen from prokaryote to eukaryote, confers negative charges on the cell surface and seems to be functionally similar to sialylation, which is found at the end of human-type complex oligosaccharide. However, detailed molecular mechanisms underlying pyruvylation have not been clarified well. Here, we first determined the crystal structure of fission yeast pyruvyltransferase Pvg1p at a resolution of 2.46 Å. Subsequently, by combining molecular modeling with mutational analysis of active site residues, we obtained a Pvg1p mutant (Pvg1p(H168C)) that efficiently transferred pyruvyl moiety onto a human-type complex glycopeptide. The resultant pyruvylated human-type complex glycopeptide recognized similar lectins on lectin arrays as the α2,6-sialyl glycopeptides. This newly-generated pyruvylation of human-type complex oligosaccharides would provide a novel method for glyco-bioengineering.
Assuntos
Aldeído-Cetona Transferases/química , Aldeído-Cetona Transferases/genética , Oligossacarídeos/química , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Engenharia de Proteínas/métodos , Dobramento de Proteína , Piruvatos/química , Schizosaccharomyces/genética , Especificidade por SubstratoRESUMO
α-Ketoacid dehydrogenases are large multi-enzyme machineries that orchestrate the oxidative decarboxylation of α-ketoacids with the concomitant production of acyl-CoA and NADH. The first reaction, catalysed by α-ketoacid decarboxylases (E1 enzymes), needs a thiamine diphosphate cofactor and represents the overall rate-limiting step. Although the catalytic cycles of E1 from the pyruvate dehydrogenase (E1p) and branched-chain α-ketoacid dehydrogenase (E1b) complexes have been elucidated, little structural information is available on E1o, the first component of the α-ketoglutarate dehydrogenase complex, despite the central role of this complex at the branching point between the TCA (tricarboxylic acid) cycle and glutamate metabolism. In the present study, we provide structural evidence that MsKGD, the E1o (α-ketoglutarate decarboxylase) from Mycobacterium smegmatis, shows two conformations of the post-decarboxylation intermediate, each one associated with a distinct enzyme state. We also provide an overall picture of the catalytic cycle, reconstructed by either crystallographic snapshots or modelling. The results of the present study show that the conformational change leading the enzyme from the initial (early) to the late state, although not required for decarboxylation, plays an essential role in catalysis and possibly in the regulation of mycobacterial E1o.
Assuntos
Proteínas de Bactérias/metabolismo , Carboxiliases/metabolismo , Modelos Moleculares , Mycobacterium smegmatis/enzimologia , Processamento de Proteína Pós-Traducional , Redobramento de Proteína , Adipatos/química , Adipatos/metabolismo , Aldeído-Cetona Transferases/química , Aldeído-Cetona Transferases/genética , Aldeído-Cetona Transferases/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Carboxiliases/química , Carboxiliases/genética , Domínio Catalítico , Descarboxilação , Complexo Cetoglutarato Desidrogenase/química , Complexo Cetoglutarato Desidrogenase/genética , Complexo Cetoglutarato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Conformação Molecular , Simulação de Acoplamento Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Acetohydroxyacid synthase (AHAS), a potential target for antimicrobial agents, catalyzes the first common step in the biosynthesis of the branched-chain amino acids. The genes of both catalytic and regulatory subunits of AHAS from Bacillus anthracis (Bantx), a causative agent of anthrax, were cloned, overexpressed in Escherichia coli, and purified to homogeneity. To develop novel anti-anthracis drugs that inhibit AHAS, a chemical library was screened, and four chemicals, AVS2087, AVS2093, AVS2387, and AVS2236, were identified as potent inhibitors of catalytic subunit with IC(50) values of 1.0 +/- 0.02, 1.0 +/- 0.04, 2.1 +/- 0.12, and 2.0 +/- 0.08 microM, respectively. Further, these four chemicals also showed strong inhibition against reconstituted AHAS with IC(50) values of 0.05 +/- 0.002, 0.153 +/- 0.004, 1.30 +/- 0.10, and 1.29 +/- 0.40 microM, respectively. The basic scaffold of the AVS group consists of 1-pyrimidine-2-yl-1H-[1,2,4]triazole-3-sulfonamide. The potent inhibitor, AVS2093 showed the lowest binding energy, -8.52 kcal/mol and formed a single hydrogen bond with a distance of 1.973 A. As the need for novel antibiotic classes to combat bacterial drug resistance increases, the screening of new compounds that act against Bantx-AHAS shows that AHAS is a good target for new anti-anthracis drugs.
Assuntos
Aldeído-Cetona Transferases/antagonistas & inibidores , Aldeído-Cetona Transferases/química , Antibacterianos/química , Bacillus anthracis/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Inibidores Enzimáticos/química , Pirimidinas/química , Aldeído-Cetona Transferases/genética , Aldeído-Cetona Transferases/metabolismo , Antraz/tratamento farmacológico , Antraz/enzimologia , Antibacterianos/uso terapêutico , Domínio Catalítico , Inibidores Enzimáticos/uso terapêutico , Ligação de Hidrogênio , Ligação Proteica , Pirimidinas/uso terapêutico , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Dihydroxyacetone synthase (DHAS) is a key enzyme involved in the assimilation of methanol in Mycobacterium sp. strain JC1 DSM 3803. The structural gene encoding DHAS in Mycobacterium sp. strain JC1 was cloned using random-primed probes synthesized after PCR with synthetic primers based on the amino acid sequences conserved in two yeast DHASs and several transketolases. The cloned gene, dasS, had an ORF of 2193 nt, encoding a protein with a calculated molecular mass of 78,197 Da. The deduced amino acid sequence of dasS contained an internal sequence of Mycobacterium sp. strain JC1 DHAS and exhibited 29.2 and 27.3 % identity with those of Candida boidinii and Hansenula polymorpha enzymes, respectively. Escherichia coli transformed with the cloned gene produced a novel protein with a molecular mass of approximately 78 kDa, which cross-reacted with anti-DHAS antiserum and exhibited DHAS activity. Primer-extension analysis revealed that the transcriptional start site of the gene was the nucleotide A located 31 bp upstream from the dasS start codon. RT-PCR showed that dasS was transcribed as a monocistronic message. Northern hybridization and beta-galactosidase assay with the putative promoter region of dasS revealed that the gene was transcribed only in cells growing on methanol. The expression of dasS in Mycobacterium sp. strain JC1 was free from catabolite repression.
Assuntos
Aldeído-Cetona Transferases , Clonagem Molecular , Mycobacterium/enzimologia , Aldeído-Cetona Transferases/química , Aldeído-Cetona Transferases/genética , Aldeído-Cetona Transferases/metabolismo , Meios de Cultura/química , Primers do DNA , Evolução Molecular , Metanol/metabolismo , Dados de Sequência Molecular , Mycobacterium/genética , Mycobacterium/crescimento & desenvolvimento , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Transcrição GênicaRESUMO
Enzymes are increasingly being used to perform regio- and enantioselective reactions in chemoenzymatic syntheses. To utilize enzymes for unphysiological reactions and to yield novel products, a broad substrate spectrum is desirable. Thiamin diphosphate (ThDP)-dependent enzymes vary in their substrate tolerance from rather strict substrate specificity (phosphoketolases, glyoxylate carboligase) to more permissive enzymes (transketolase, dihydroxyacetone synthase, pyruvate decarboxylase) and therefore differ in their potential to be used as biocatalysts. We give an overview of the known substrate spectra of ThDP-dependent enzymes and present examples of multi-enzyme or chemoenzymatic approaches which involve ThDP-dependent enzymes as biocatalysts to obtain pharmaceutical compounds as ephedrine and glycosidase inhibitors, sex pheromones as exo-brevicomin, 13C-labeled metabolites, and other intermediates as 1-deoxyxylulose 5-phosphate, a precursor of vitamins and isoprenoids.