RESUMEN
A methyl parathion hydrolase (MPH) gene, bjmpd, was cloned from a newly isolated MP-degrading bacterial strain, Burkholderia jiangsuensis MP-1T and heterologously expressed in Escherichia coli BL21 (DE3). Although the amino acid sequence of the bjmpd-encoded enzyme, named BjMPH, differed from that of MPH from Pseudomonas sp. WBC-3 (PsMPH) in only three residues, Ser132, Val247 and Ala267, a significantly higher specific activity towards MP was exhibited by BjMPH than PsMPH. Among them, Ala267 was identified as a key site affecting the catalytic efficiency, and it was rather conservative (Ala or Ser) in homologous proteins, suggesting that a simple substitution of the residue in conservative site with another conservative residue based on the consensus sequence approach might possibly enhance the catalytic efficiency of the MP-degrading enzyme. Inspired by such an observation, we identified a new mutant, BjMPHT64N, exhibiting 3.78-fold higher catalytic efficiency (kcat/KM) towards MP than its wild-type, reaching 4.20×106M-1s-1. The mutant BjMPHT64N also displayed enhanced reactivities (kcat/KM) towards other organophosphorus pesticides. Homology-modelling analysis indicates that enhanced polar contacts of the 64th residue in this mutant may contribute to stabilizing the structure of the enzyme and promote the interactions between enzyme and substrate. This study generated an efficient MP-degrading enzyme, and provides useful information for enhancing the catalytic efficiency of MPHs via conservative residue substitution based on the consensus approach.
Asunto(s)
Proteínas Bacterianas/metabolismo , Metil Paratión/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biodegradación Ambiental , Burkholderia/enzimología , Burkholderia/genética , Dominio Catalítico/genética , Secuencia de Consenso , Secuencia Conservada , Evolución Molecular Dirigida , Estabilidad de Enzimas , Genes Bacterianos , Enlace de Hidrógeno , Insecticidas/metabolismo , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología Estructural de ProteínaRESUMEN
A methyl parathion (MP) degrading bacterial strain, designated MP-1(T), was isolated from a waste land where pesticides were formerly manufactured in Jiangsu province, China. Polyphasic taxonomic studies showed that MP-1(T) is a Gram-stain-negative, non-spore-forming, rod-shaped and motile bacterium. The bacterium could grow at salinities of 0-1â% (w/v) and temperatures of 15-40 °C. Strain MP-1(T) could reduce nitrate to nitrite, utilize d-glucose and l-arabinose, but not produce indole, or hydrolyse gelatin. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that MP-1(T) belongs to the genus Burkholderia, showing highest sequence similarity to Burkholderia grimmiae DSM 25160(T) (98.5â%), and similar strains including Burkholderia zhejiangensis OP-1(T) (98.2â%), Burkholderia choica LMG 22940(T) (97.5â%), Burkholderia glathei DSM 50014(T) (97.4â%), Burkholderia terrestris LMG 22937(T) (97.2â%) and Burkholderia telluris LMG 22936(T) (97.0â%). In addition, the gyrB and recA gene segments of strain MP-1(T) exhibited less than 89.0â% and 95.1â% similarities with the most highly-related type strains indicated above. The G+C content of strain MP-1(T) was 62.6 mol%. The major isoprenoid quinone was ubiquinone Q-8. The predominant polar lipids comprised phosphatidyl ethanolamine, phosphatidyl glycerol, aminolipid and phospholipid. The principal fatty acids in strain MP-1(T) were C18â:â1ω7c/C18â:â1ω6c (23.3â%), C16â:â0 (16.8â%), cyclo-C17â:â0 (15.0â%), C16â:â1ω7c/C16â:â1ω6 (8.5â%), cyclo-C19â:â0ω8c (8.1â%), C16â:â1 iso I/C14â:â0 3-OH (5.7â%), C16â:â0 3-OH (5.6â%) and C16â:â02-OH (5.1â%). The DNA-DNA relatedness values between strain MP-1(T) and the three type strains (B. grimmiae DSM 25160(T), B. zhejiangensis OP-1(T) and B. glathei DSM 50014(T)) ranged from 24.6â% to 37.4â%. In accordance with phenotypic and genotypic characteristics, strain MP-1(T) represents a novel species of the genus Burkholderia, for which the name Burkholderia jiangsuensis sp. nov. is proposed, the type strain is MP-1(T) (LMG 27927(T)â=âMCCC 1K00250(T)).
Asunto(s)
Burkholderia/clasificación , Metil Paratión/metabolismo , Filogenia , Microbiología del Suelo , Técnicas de Tipificación Bacteriana , Composición de Base , Burkholderia/genética , Burkholderia/aislamiento & purificación , China , ADN Bacteriano/genética , Ácidos Grasos/química , Insecticidas/metabolismo , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Fosfolípidos/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Contaminantes del Suelo/metabolismo , Ubiquinona/químicaRESUMEN
Burkholderia sp. strain MP-1 was isolated from pesticide-contaminated soil. Herein, we report the draft genome sequence of strain MP-1, which contains 168 contigs of 8,611,053 bp, with a G+C content of 62.55% and 7,631 protein-coding genes.
