Crystallization and preliminary X-ray analysis of the reductase component of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii

Abstract

p-Hydroxyphenylacetate 3-hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) at the ortho position to yield 3,4-dihydroxyphenylacetate (DHPA). HPAH from A. baumannii is a two-component flavoprotein consisting of a smaller reductase (C(1)) component and a larger oxygenase (C(2)) component. The C(1) component supplies a reduced flavin in its free form to the C(2) counterpart for hydroxylation. In addition, HPA can bind to C(1) and enhance the flavin-reduction rate without becoming hydroxylated. The recombinant C(1) component was purified and crystallized using the microbatch method at 295 K. X-ray diffraction data were collected to 2.3 Å resolution using synchrotron radiation on the BL13B1 beamline at NSRRC, Taiwan. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 47.78, b = 59.92, c = 211.85 Å, and contained two molecules of C(1) per asymmetric unit.

Figure 1

A crystal of the C₁ component of A. baumannii HPAH. This orthorhombic crystal grew to dimensions of 0.15 × 0.15 × 0.5 mm in 3 d.

Figure 2

X-ray diffraction pattern of the C₁ crystal. The crystal was mounted such that its long axis was nearly parallel to the spindle axis to minimize reflection overlap. This was accomplished by the use of an L-shaped nylon loop (inset). While maintaining the crystal-to-detector distance of 400 mm to prevent spot overlaps, the CCD detector was offset to allow the collection of higher resolution data at the upper side of the detector.