Molecular cloning, heterologous expression, and primary structure of the structural gene for the copper enzyme nitrous oxide reductase from denitrifying Pseudomonas stutzeri.
AUTOR(ES)
Viebrock, A
RESUMO
The nos genes of Pseudomonas stutzeri are required for the anaerobic respiration of nitrous oxide, which is part of the overall denitrification process. A nos-coding region of ca. 8 kilobases was cloned by plasmid integration and excision. It comprised nosZ, the structural gene for the copper-containing enzyme nitrous oxide reductase, genes for copper chromophore biosynthesis, and a supposed regulatory region. The location of the nosZ gene and its transcriptional direction were identified by using a series of constructs to transform Escherichia coli and express nitrous oxide reductase in the heterologous background. Plasmid pAV5021 led to a nearly 12-fold overexpression of the NosZ protein compared with that in the P. stutzeri wild type. The complete sequence of the nosZ gene, comprising 1,914 nucleotides, together with 282 nucleotides of 5'-flanking sequences and 238 nucleotides of 3'-flanking sequences was determined. An open reading frame coded for a protein of 638 residues (Mr, 70,822) including a presumed signal sequence of 35 residues for protein export. The presequence is in conformity with the periplasmic location of the enzyme. Another open reading frame of 2,097 nucleotides, in the opposite transcriptional direction to that of nosZ, was excluded by several criteria from representing the coding region for nitrous oxide reductase. Codon usage for nosZ of P. stutzeri showed a high G + C content in the degenerate codon position (83.9% versus an average of 60.2%) and relaxed codon usage for the Glu codon, characteristic features of Pseudomonas genes from other species. E. coli nitrous oxide reductase was purified to homogeneity. It had the Mr of the P. stutzeri enzyme but lacked the copper chromophore.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=211505Documentos Relacionados
- NosR, a membrane-bound regulatory component necessary for expression of nitrous oxide reductase in denitrifying Pseudomonas stutzeri.
- Operon Structure and Regulation of the nos Gene Region of Pseudomonas stutzeri, Encoding an ABC-Type ATPase for Maturation of Nitrous Oxide Reductase
- Physical mapping of transposon Tn5 insertions defines a gene cluster functional in nitrous oxide respiration by Pseudomonas stutzeri.
- Formation of the N-N bond from nitric oxide by a membrane-bound cytochrome bc complex of nitrate-respiring (denitrifying) Pseudomonas stutzeri.
- Cloning, expression, and primary structure of a Chlamydia trachomatis binding protein.