Abstract
A novel alliinase (EC 4.4.1.4) was detected and purified from the roots of the Amazonian medicinal plant Petiveria alliacea. The isolated enzyme is a heteropentameric glycoprotein composed of two alpha-subunits (68.1 kD each), one beta-subunit (56.0 kD), one gamma-subunit (24.8 kD), and one delta-subunit (13.9 kD). The two alpha-subunits are connected by a disulfide bridge, and both alpha- and beta-subunits are glycosylated. The enzyme has an isoelectric point of 4.78 and pH and temperature optima of 8.0 and approximately 52 degrees C, respectively. Its activation energy with its natural substrate S-benzyl-l-cysteine sulfoxide is 64.6 kJ mol(-1). Kinetic studies showed that both K(m) and V(max) vary as a function of substrate structure, with the most preferred substrates being the naturally occurring P. alliacea compounds S-benzyl-l-cysteine sulfoxide and S-2-hydroxyethyl-l-cysteine sulfoxide. The alliinase reacts with these substrates to produce S-benzyl phenylmethanethiosulfinate and S-(2-hydroxyethyl) 2-hydroxyethanethiosulfinate, respectively.
Highlights
A novel alliinase (EC 4.4.1.4) was detected and purified from the roots of the Amazonian medicinal plant Petiveria alliacea
Our suspicion of the presence of an alliinase in P. alliacea roots was confirmed with our successful isolation of a protein that reliably resulted in the production of the thiosulfinate petivericin on exposure to its corresponding Cys sulfoxide precursor petiveriin
During the development of the chromatographic separation protocol for the isolation and purification of this protein, it became necessary for us to design a facile and efficient means by which to confirm and track the presence of alliinase activity. We have developed such a method based on modification of a procedure previously reported by Ukai and Sekiya (1997) for the determination of lyases that cleave C-S bonds in sulfur-containing compounds after PAGE
Summary
A novel alliinase (EC 4.4.1.4) was detected and purified from the roots of the Amazonian medicinal plant Petiveria alliacea. The enzyme is compartmentalized in plant cell vacuoles, while its substrates, S-alk(en)yl-L-Cys sulfoxides (ACSOs), are located in the cytoplasm (Lancaster and Collin, 1981). It is indigenous to the Amazon rainforest and tropical areas of Central and South America, the Caribbean, and sub-Saharan Africa (Kubec and Musah, 2001; Kubec et al, 2002) It has a long history of use in herbal medicine. Many of which have been shown to have biological activity, have been isolated from P. alliacea, including benzyl 2-hydroxyethyl trisulfide (Szczepanski et al, 1972), cis-3,5-diphenyl-1,2,4-trithiolan (Adesogan, 1974), dibenzyl trisulfide (Sousa et al, 1990), dibenzyl disulfide (Ayedoun et al, 1998), S-benzyl-L-Cys sulfoxide (petiveriin), S-2-hydroxyethyl-L-Cys sulfoxide (2-hydroxyethiin), (Z)-phenylmethanethial S-oxide (PMTSO), and S-benzyl phenylmethanethiosulfinate (petivericin; Kubec and Musah, 2001; Kubec et al, 2002, 2003), among others
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