Cloning and heterologous expression of P450Um-1, a novel bacterial P450 gene, for hydroxylation of immunosuppressive agent AS1387392

Abstract

Biotransformation technology involving enzymatic modification of original substrates by organisms such as microbes is a valuable tool in improving pharmacokinetics or physicochemical properties of the base compounds. The fungal metabolite AS1387392 is a histone deacetylase inhibitor with potential as a therapeutic immunosuppressant. However, its paucity of functional groups, essential to synthesizing derivatives, is a drawback. Amycolatopsis azurea JCM-3275 catalyzed hydroxylation of AS1387392 to AS1429716, which may facilitate the synthesis of more derivatives by the additional hydroxyl moiety present in AS1429716. This reaction was inhibited by cytochrome P450 inhibitor metyrapone, indicating that cytochrome P450 may be responsible for the transformation. Degenerate PCR primers were subsequently constructed and used to clone genes encoding cytochrome P450 from the genomic DNA of A. azurea JCM-3275. We cloned an entire novel P450 gene (1209 bp) and named it P450Um-1. Its deduced amino acid sequence was homologous with that of the CYP105 subfamily. Further cloning of the upstream region, which may contain the native promoter site, was followed by insertion of the open reading frame with the upstream area into Streptomycetes high copy vector pIJ702, giving the expression plasmid pNUm-1. P450Um-1 was specifically expressed in Streptomyces lividans TK24, and this recombinant strain converted AS1387392 to AS1429716 without any redox partners. These results show that P450Um-1, a novel bacterial P450, catalyzed hydroxylation of AS1387392 to AS1429716. This resultant recombinant strain is expected to be an efficient biocatalyst with application to more suitable redox systems than those tested here.

Bioconversion of AS1387392: bioconversion studies involving Amycolatopsis azurea JCM 3275

“…In particular, suppression of hydroxyketone reduction may improve the conversion yield, and the cloning and heterologous expression of the hydroxylation enzyme may help improve production of 2. 9 The immunosuppressive activities of both 2 and 3 were as potent as 1. However, no activity was noted for 4, 5 or 6, which were formed following reduction of a hydroxyketone, indicating that a hydroxyketone is essential for HDAC inhibition.…”

Characterization of a novel cytochrome P450 from Amycolatopsis sp. CGMCC1149 for hydroxylation of lovastatin

“…). The sequencing result shows that p450lov shares 91% nucleotide identities (E‐value 0.0) with P450Um‐1 from Amycolatopsis azured .…”

AS1387392, a novel immunosuppressive cyclic tetrapeptide compound with inhibitory activity against mammalian histone deacetylase

“…Changes of the code name of the reportedly novel immunosuppressive cyclic tetra-peptide published in The Journal of Antibiotics, November issue, 63, 633-636 (2010), and the following three articles [1][2][3] were not clearly explained. In addition, citations to the synthetic preparation and testing of those compounds were not included.…”