Characterization of NucPNP and NucV involved in the early steps of nucleocidin biosynthesis in Streptomyces calvus.

Utumporn Ngivprom,Siriwalee Siriwibool,Qi Zhang,Wenjuan Ji,Surayut Kluaiphanngam,Anyanee Kamkaew,Rung-Yi Lai,James R Ketudat Cairns

doi:10.1039/d0ra10878b

Abstract

Nucleocidin 1 produced by Streptomyces calvus is one of five characterized natural products containing fluorine. It was discovered in 1956, but its biosynthesis is not yet completely resolved. Recently, the biosynthetic gene cluster of 1 was identified. The nucPNP gene, which was initially annotated as orf206 and encodes a putative purine nucleoside phosphorylase, is essential for nucleocidin production. In this study, we performed in vitro assays and showed NucPNP produced adenine 3 from methylthioadenosine (MTA) 2 and adenosine 4. We also showed the downstream enzyme, NucV annotated as adenine phosphoribosyltransferase (APRT), catalyzes AMP formation from adenine 3 and 5-phospho-α-d-ribose-1-diphosphate (PRPP) 5. However, the catalytic efficiency of NucV was much slower than its homolog ScAPRT involved in the biosynthesis of canonical purine nucleoside in the same strain. These results provide new insights in nucleocidin biosynthesis and could guide future research on organofluorine formation.

Highlights

Organo uorine compounds play important roles in pharmaceuticals,[1] medical imaging,[2] materials science,[3] agrochemicals,[4] and other disciplines
3.1 NucPNP catalysis of phosphorylation of purine nucleoside NucPNP was annotated as purine nucleoside phosphorylase (PNP).[18]
PNP enzymes catalyze a reversible reaction in purine metabolism,[21] converting purine nucleoside and phosphate to purine base and a-D-ribose-1-phosphate

Summary

Introduction

Organo uorine compounds play important roles in pharmaceuticals,[1] medical imaging,[2] materials science,[3] agrochemicals,[4] and other disciplines. NucV, which encodes an adenine phosphoribosyltransferase (APRT), showed a increasing level of mRNA transcript (110 times) as nucPNP. NucV catalyzes the synthesis of AMP 6 from adenine 3 and PRPP 5, but its rate was slower than ScAPRT Based on these ndings, the biosynthetic pathway is proposed in Scheme 1, which is similar to the biosynthesis of ascamycin and dealanylascamycin.[19] The substrate of NucV is proposed as 40-F-PRPP 7 from the NucJ reaction. These results provide clues to solve the mystery of C–F formation in nucleocidin biosynthesis

Materials

Enzymatic assays

Kinetic constant determination

Results and discussion

The NucV-catalyzed AMP formation is slower than those by ScAPRT and EcAPRT

Conclusions and perspective