Purification and crystallographic analysis of a FAD-dependent halogenase from Streptomyces sp. JCM9888

Abstract

A new FAD (flavin adenine dinucleotide)-dependent halogenase HalY from Streptomyces sp. JCM9888 was reported to be involved in the regioselective halogenation of adenine. HalY is a variant B FAD-dependent halogenase that is most similar to the halogenase PltA involved in pyoluteorin biosynthesis. This study reports the overexpression and purification of HalY with an N-terminal hexahistidine tag, followed by crystallization experiments and X-ray crystallographic analysis. HalY was purified as a monomer in solution and crystallized to give X-ray diffraction to a resolution of 1.7 Å. The crystal belonged to the monoclinic space group P21, with unit-cell parameters a = 41.4, b = 113.4, c = 47.6 Å, α = γ = 90, β = 107.4°, and contained one monomer of HalY in the asymmetric unit, with a calculated Matthews coefficient of 2.3 Å(3) Da(-1) and a solvent content of 46%. The structure of the halogenase CndH was used as a search model in molecular replacement to obtain the initial model of HalY. Manual model building and structure refinement of HalY are in progress.

Keywords: FAD-dependent halogenase; adenine; regioselective halogenation.

Figure 1

Multiple sequence alignment of the FAD-dependent halogenases from Streptomyces sp. JCM9888 against the homologous halogenases from the biosynthetic pathways of pyoluteorin (PltA, AAY92059.1), chondrochloren (CndH, Q7S6N4) and chloramphenicol (CmlS, Q9AL91).

Figure 2

(a) Purification profile of HalY from size-exclusion chromatography. The solid curve shows the UV absorption at 280 nm and the elution volume for the major peak of HalY is labelled. (b) SDS–PAGE of purified HalY fractions from size-exclusion chromatography; the middle three fractions were used for crystallization. Lane 1 shows molecular-weight markers labelled in kDa.

Figure 3

(a) Crystals of HalY grown in 0.1 M bis-tris propane pH 6.3, 16%(w/v) PEG 3350, 0.2 M NaCl, which were used in X-ray diffraction data collection. (b) Diffraction image of HalY on BSRF beamline 3W1A. The circle indicates 1.70 Å resolution.