Functional expression and purification of DoxA, a key cytochrome P450 from Streptomyces peucetius ATCC 27952

PeerJ. 2022 Nov 16:10:e14373. doi: 10.7717/peerj.14373. eCollection 2022.

Abstract

The antitumor drug doxorubicin is widely used in clinical practice. However, the low yield and high cost of this drug highlight the urgent need for cost-effective processes to rapidly manufacture antitumor drugs at scale. In the biosynthesis pathway, the multi-functional cytochrome P450 enzyme DoxA catalyzes the last three steps of hydroxylation. The final conversion of daunorubicin to doxorubicin is the rate-limiting step. In our work, the DoxA has been expressed with the ferredoxin reductase FDR2 and the ferredoxin FDX1 and purified to homogeneous. The reduced carbon monoxide difference spectroscopy, heme concentration, and enzymatic characteristic were characterized. These studies suggest an approach for engineering Streptomyces P450s with functional expression for mechanistic and structural studies.

Keywords: Cytochrome P450; DoxA; Doxorubicin synthase; Functional expression; Purification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cytochrome P-450 Enzyme System / genetics
  • Doxorubicin
  • Ferredoxins* / metabolism
  • Streptomyces* / genetics

Substances

  • Ferredoxins
  • Cytochrome P-450 Enzyme System
  • Doxorubicin

Supplementary concepts

  • Streptomyces peucetius

Grants and funding

This work was supported by the Natural Science Foundation of Guangxi Province (Grant No. 2018GXNSFAA281019 and 2022GXNSFBA035536), and the National Natural Science Foundation of China (Grant No. 31860245 and 31960203). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.