Cloning and characterization of the genes encoding nitrilotriacetate monooxygenase of Chelatobacter heintzii ATCC 29600

Abstract

A 6.2-kb DNA fragment containing the genes for the nitrilotriacetate (NTA) monooxygenase of Chelatobacter heintzii ATCC 29600 was cloned and characterized by DNA sequencing and expression studies. The nucleotide sequence contained three major open reading frames (ORFs). Two of the ORFs, which were oriented divergently with an intergenic region of 307 bp, could be assigned to the NTA monooxygenase components A and B. The predicted N-terminal amino acid sequences of these ORFs were identical with those determined for the purified components. We therefore named these genes ntaA (for component A of NTA monooxygenase) and ntaB (for component B). The ntaA and ntaB genes could be expressed in Escherichia coli DH5␣, and the gene products were visualized after Western blotting (immunoblotting) and incubation with polyclonal antibodies against component A or B. By mixing overproduced NtaB from E. coli and purified component A from C. heintzii ATCC 29600, reconstitution of a functional NTA monooxygenase complex was possible. The deduced gene product of ntaA showed only significant homology to SoxA (involved in dibenzothiophene degradation) and to SnaA (involved in pristamycin synthesis); that of ntaB shared weak homologies in one domain with other NADH:flavine mononucleotide oxidoreductases. These homologies provide no conclusive answer as to the possible evolutionary origin of the NTA monooxygenase. The deduced gene product of the third ORF (ORF1) had homology in the N-terminal region with the GntR class of bacterial regulator proteins and therefore may encode a regulator protein, possibly involved in regulation of ntaA and ntaB expression.

Identification and characterization of the two-enzyme system catalyzing oxidation of EDTA in the EDTA-degrading bacterial strain DSM 9103

“…The NH 2 -terminal sequences of cA (22) and cAЈ showed no significant similarities. Moreover, immunoblotting with polyclonal antibodies against cA and cB did not reveal further similarities at the protein level between the two equivalent components of both enzyme complexes.…”

Functional Analysis of the Small Component of the 4-Hydroxyphenylacetate 3-Monooxygenase of Escherichia coli W: a Prototype of a New Flavin:NAD(P)H Reductase Subfamily

“…The genes encoding the two components of the enzymes of the TC-FDM family are located in the same operon or can be found very close in the chromosome (32), sometimes divergently oriented (24). Such an arrangement could favor a coordinated regulation of both genes and might facilitate the horizontal transfer of the monooxygenase activity.…”

Bacillus subtilis genes for the utilization of sulfur from aliphatic sulfonates

“…Since we have not tested whether disruption of ssuD results in the same phenotype as disruption of ssuA and ssuC, it cannot be excluded that SsuD is the only enzyme that is able to degrade sulfonates. The proteins which are related in sequence to SsuD, SnaA from Streptomyces pristinaespiralis, NtaA from Chelatobacter heintzii and Ssi6 from E. coli, require FMNH, for activity (Blanc et al, 1995;Knobel et al, 1996; E. Eichhorn, J. R. van der Ploeg & T. Leisinger, unpublished). The FMNH, is provided by NADH : : FMN oxidoreductases, for which the corresponding genes are located in the same gene cluster as the genes encoding the monooxygenase.…”