Crystallization and preliminary X-ray characterization of the Bacillus amyloliquefaciens YwrO enzyme

Abstract

CB1954 is an anticancer prodrug that is currently in clinical trials coupled with the Escherichia coli flavoenzyme nitroreductase (NTR) for use in directed-enzyme prodrug therapy (DEPT). The NTR enzyme is responsible for the conversion of the prodrug into a cytotoxic agent. The bifunctional alkylating agent produced by this bioactivation process leads to DNA damage and death of cancer cells. Recently, a novel flavoenzyme from Bacillus amyloliquefaciens, YwrO (Bam YwrO), was reported to be able to reduce CB1954 from its noncytotoxic form into its active form. The crystallization and preliminary X-ray diffraction analysis of two crystal forms of Bam YwrO are reported. The first crystal form is orthorhombic, with space group P22(1)2(1), and diffracts X-rays to 2.18 A resolution. The second crystal form is tetragonal, with space group P4(1), and diffracts X-rays to 3.4 A. Determination of the Bam YwrO crystal structure will provide an understanding of the molecular recognition between this enzyme and the anticancer prodrug CB1954.

Figure 1

The four-electron bioactivation mechanism of CB1954 by NQO2 (Knox & Chen, 2004 ▶).

Figure 2

Bam YwrO crystals grown from (a) 67.5% MPD and 0.1 M bicine pH 9 (crystal form 1, orthorhombic) and (b) 2.4 M ammonium sulfate and 0.1 M citric acid pH 4 (crystal form 2, tetragonal). The crystals were grown using the vapour-diffusion technique with sitting drops in 24-well plates.

Figure 3

Diffraction patterns obtained from (a) crystal form 1 (orthorhombic) and (b) crystal form 2 (tetragonal). Resolution ranges are shown (values are in angstroms).