Purification, characterization, and crystallization of alliinase from garlic

Abstract

Glycosylated dimeric alliinase (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 α-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 12 12 1, a=68.4, b=101.1, c=155.7 Å) grown from an ammonium sulfate solution. These crystals diffract to at least 1.5 Å resolution at a synchrotron source and are suitable for structure determination.