Crystallization and preliminary X-ray analysis of AAMS amidohydrolase, the final enzyme in degradation pathway I of pyridoxine

Abstract

alpha-(N-Acetylaminomethylene)succinic acid (AAMS) amidohydrolase from Mesorhizobium loti MAFF303099, which is involved in a degradation pathway of vitamin B(6) and catalyzes the degradation of AAMS to acetic acid, ammonia, carbon dioxide and succinic semialdehyde, has been overexpressed in Escherichia coli. To elucidate the reaction mechanism based on the tertiary structure, the recombinant enzyme was purified and crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as precipitant. A crystal of the enzyme belonged to the monoclinic space group C2, with unit-cell parameters a = 393.2, b = 58.3, c = 98.9 A, beta = 103.4 degrees , and diffraction data were collected to 2.7 A resolution. The V(M) value and calculation of the self-rotation function suggested that three dimers with a threefold symmetry were possibly present in the asymmetric unit.

Figure 1

The enzyme reaction catalyzed by AAMS amidohydrolase.

Figure 2

SDS–PAGE analysis of purified AAMS amidohydrolase. Lane 1, molecular-weight markers. Lane 2, purified AAMS amidohydrolase for crystallization.

Figure 3

Crystal of M. loti AAMS amidohydrolase with dimensions of 0.14 × 0.06 × 0.01 mm.

Figure 4

Diffraction image of M. loti AAMS amidohydrolase; the ring indicates 2.7 Å resolution.