Purification, characterization, and crystallization of alliinase from garlic

Glycosylated dimeric allimase (EC 4.4.1.4) was purified to homogeneity from its natural source, garlic. With 660 units/mg, the specific enzymatic activity of the pure enzyme is the highest reported to date. Based on both CD spectroscopy data and sequence-derived secondary structure prediction, the alpha-helix content of alliinase was estimated to be about 30%. Comparisons of all available amino acid sequences of alliinases revealed a common cysteine pattern of the type C-x(18-19)-C-X-C-x(2)-C-x(5)-C-x(6)-C in the N-terminal part of the sequences. This pattern is conserved in alliinases but absent in other pyridoxal 5'-phosphate-dependent enzymes. It suggests the presence of an epidermal growth factor-like domain in the three-dimensional structures of alliinases, making them unique among the various families of pyridoxal 5-phosphate-dependent enzymes. Well-ordered three-dimensional crystals of garlic alliinase were obtained in four different forms. The best diffraction was observed with crystal form IV (space group P2(1)2(1)2(1), a = 68.4, b = 101.1, c = 155.7 Angstrom) grown from an ammonium sulfate solution. These crystals diffract to at least 1.5Angstrom resolution at a synchrotron source and are suitable for structure determination. (C) 2002 Elsevier Science (USA). All rights reserved.