Characterization of the P450 Monooxygenase NysL, Responsible for C-10 Hydroxylation during Biosynthesis of the Polyene Macrolide Antibiotic Nystatin in Streptomyces noursei

Abstract

The nysL gene, encoding a putative P450 monooxygenase, was identified in the nystatin biosynthetic gene cluster of Streptomyces noursei. Although it has been proposed that NysL is responsible for hydroxylation of the nystatin precursor, experimental evidence for this activity was lacking. The nysL gene was inactivated in S. noursei by gene replacement, and the resulting mutant was shown to produce 10-deoxynystatin. Purification and an in vitro activity assay for 10-deoxynystatin demonstrated its antifungal activity being equal to that of nystatin. The NysL protein was expressed heterologously in Escherichia coli as a His-tagged protein and used in an enzyme assay with 10-deoxynystatin as a substrate. The results obtained clearly demonstrated that NysL is a hydroxylase responsible for the post-polyketide synthase modification of 10-deoxynystatin at position C-10. Kinetic studies with the purified recombinant enzyme allowed determination of K m and k cat and revealed no inhibition of recombinant NysL by either the substrate or the product. These studies open the possibility for in vitro evolution of NysL aimed at changing its specificity, thereby providing new opportunities for engineered biosynthesis of novel nystatin analogues hydroxylated at alternative positions of the macrolactone ring.

New Nystatin-Related Antifungal Polyene Macrolides with Altered Polyol Region Generated via Biosynthetic Engineering of Streptomyces noursei

“…The MIC value of BSG022 was similar to that of S44HP ( Table 3), indicating that the C-10 hydroxyl removal had no significant effect on the antifungal activity, which was in agreement with the previous report on 10-deoxynystatin (23). The HC 50 value of BSG022 was more than 2-fold reduced compared to that of S44HP, suggesting increased toxicity to erythrocytes.…”

“…In order to determine whether the effect on biological activity observed for the BSG003 and BSG018 molecules was due to the changes at C-9 or C-10, we decided to remove a single C-10 hydroxyl from the S44HP and BSG005 analogues. In a previous report we demonstrated that inactivation of the P450 mono-oxygenase gene nysL in S. noursei resulted in a mutant strain, designated NLD101, which produced 10-deoxynystatin with antifungal activity similar to that of the parental antibiotic nystatin (23). However, the hemolytic activity of 10-deoxynystatin has not been tested.…”

Selvamicin, an atypical antifungal polyene from two alternative genomic contexts

“…SelL, a cytochrome p450 with homology to the p450 NysL that installs nystatin's C10 hydroxyl, is the most probable oxidase for C4 (38). We also identified SelP, a 2-oxoglutarate-dependent oxygenase with homology to phytanoylCoA dioxygenases.…”

Versatility of Enzymes Catalyzing Late Steps in Polyene 67-121C Biosynthesis

“…This construct was introduced into S. nodosus, and polyenes were extracted and analyzed by HPLC. The main products were amphotericins A (7) and B (3). No extra polyene species were detected as convincing peaks in the chromatograms, but ESMS analysis of the crude polyenes revealed additional trace ions with molecular masses appropriate for mannosyl-amphotericin A (10) …”

“…In some cases, another P450 subsequently introduces a hydroxyl group or epoxide. 3,4) Chemical modification studies have shown that adding sugar residues to polyenes can improve their pharmacological properties. [5][6][7] Enzymatic methods of extending natural product glycosylation have been developed in recent years.…”