Cloning sequencing and expression of the gene for cytochrome P450meg, the steroid-15β-monooxygenase from Bacillus megaterium ATCC 13368

Abstract

A 4.3 kb EcoRI fragment carrying the gene for cytochrome P450meg, the steroid-15 beta-monooxygenase from Bacillus megaterium ATCC 13368, was cloned and completely sequenced. The gene codes for a protein of 410 amino acids and was expressed in Escherichia coli and B. subtilis. Protein extracts from the recombinant E. coli strains were able to hydroxylate corticosteroids in the 15 beta position when supplemented with an extract from a P450- mutant of B. megaterium ATCC 13368 as a source of megaredoxin and megaredoxin reductase. In contrast, 15 beta-hydroxylation was obtained in vitro and in vivo without the addition of external electron transfer proteins, when cytochrome P450meg was produced in B. subtilis 168. Protein extracts from nonrecombinant B. subtilis 168 could also support the in vitro hydroxylation by cytochrome P450meg produced in E. coli.

Identification of CYP106A2 as a Regioselective Allylic Bacterial Diterpene Hydroxylase

“…A knockout strain of Bacillus megaterium ATCC 13 368 lacking the capacity to synthesize CYP106A2 served as a negative control. [13] No conversion occurred with the knockout strain, indicating that the reaction was catalyzed in the whole-cell approach by CYP106A2 ( Figure 5). Interestingly, 12b-hydroxyabietic acid was built up to a higher content than in the in vitro reaction.…”

“…[13] All experiments were carried out in a complex medium consisting of yeast extract (Difco, Detroit, MI, 24 g), glycerine (4 mL), soytone (Difco, 12 g), KH 2 PO 4 (2.31 g), K 2 HPO 4 (12.5 g), and distilled H 2 O (1000 mL). B. megaterium was stored on nutrient agar (Difco) plates at 4 8C, which in the case of the knockout strain contained tetracycline (12.5 mg L…”

“…The transfer of electrons can, however, be achieved with different electron donor proteins. [11,13,14] In order to enlarge the substrate spectrum of this unique P450, with its capacity for selective hydroxylation of complex steroidal molecules, to other compounds, we decided to apply a high-throughput substrate screening for this enzyme.…”

Theoretical and Experimental Evaluation of a CYP106A2 Low Homology Model and Production of Mutants with Changed Activity and Selectivity of Hydroxylation

“…The amino acid sequence of CYP106A2 was taken from Rauschenbach et al [53] The five template structures, namely CYP119 from Sulfolobus solfataricus (P450sulso, 1F4T), [42] CYP107A1 from Saccharopolyspora erythrea (P450eryF, 1OXA), [54] CYP108 from Pseudomonas sp. (P450terp, 1CPT), [55] CYP101 from Pseudomonas putida (P450cam, 1PHG) [56] and CYP55A1 from Fusarium oxysporum (P450nor, 1L6) [57] were obtained from the Protein Data Bank.…”

A 53-base-pair inverted repeat negatively regulates expression of the adjacent and divergently oriented cytochrome P450(BM-1) gene and its regulatory gene, bm1P1, in Bacillus megaterium

“…Immediately upstream of the P450 BM-3 structural gene exists the bm3R1 gene, which encodes a transcriptional repressor that negatively regulates the expression of the P450 BM-3 gene (18). P450 BM-1 shows a high degree of sequence similarity (63% identity, 76.5% similarity) to P450 meg , a steroid-15␤-monooxygenase isolated from Bacillus megaterium ATCC 13368 (13). Upstream of and opposite in orientation to the P450 BM-1 gene exists the bm1P1 gene, which encodes a putative positive regulator probably involved in barbiturate-mediated induction of P450 BM-1 expression (4).…”