Title
Purification, characterization, and crystallization of alliinase from garlic
Author
E. Bartholomeus Kuettner, Rolf Hilgenfeld, Manfred S. Weiss
Year
2002
Journal
Archives of Biochemistry and Biophysics
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 Full-size image (<1 K) 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 P212121,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.
Instrument
J-710
Keywords
Circular dichroism, Secondary structure, Biochemistry