Molecular cloning, nucleotide sequencing and expression of genes encoding a cytochrome P450 system involved in secondary amine utilization in Mycobacterium sp. strain RP1.

Mycobacterium sp. strain RP1 degrades morpholine, piperidine, and pyrrolidine and is able to use these compounds as the sole source of carbon, nitrogen, and energy. Cytochrome P450 (MorA) is involved in the biodegradation of these secondary amines. A 3.9-PstI genomic DNA fragment, containing the gene encoding MorA, was cloned and sequenced. Four open reading frames were detected on this DNA fragment. The first encoded a cytochrome P450 designated as MorA which was the second member of the CYP151 family and was named CYP151A2. The second open reading frame (morB) featured a [3Fe-4S] type of ferredoxin. A third gene (morC), exhibiting sequence identity to known reductases, and a fourth truncated gene encoding a putative glutamine reductase (orf1' ), were found downstream of morB. Recombinant MorA cytochrome P450 was purified to homogeneity from Escherichia coli. The purified enzyme was a monomeric soluble protein with an apparent Mr of about 45,000. CYP151A2 catalyzed the ring cleavage of the secondary amines and the Vmax/KMapp values indicated that pyrrolidine is the preferred substrate for this monooxygenase.