Studies of a Novel Cysteine Sulfoxide Lyase fromPetiveria alliacea: The First Heteromeric Alliinase

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 a-subunits (68.1 kD each), one b-subunit (56.0 kD), one g-subunit (24.8 kD), and one d-subunit (13.9 kD). The two a-subunits are connected by a disulfide bridge, and both a-and b-subunits are glycosylated. The enzyme has an isoelectric point of 4.78 and pH and temperature optima of 8.0 and approximately 52°C, respectively. Its activation energy with its natural substrate S-benzyl-L-cysteine sulfoxide is 64.6 kJ mol 21 . 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.

Discovery and Characterization of a Novel Lachrymatory Factor Synthase inPetiveria alliaceaand Its Influence on Alliinase-Mediated Formation of Biologically Active Organosulfur Compounds

“…It is well documented that glycoproteins as well as proteins with rigid disulfide linkages often exhibit anomalous migration profiles in SDS-PAGE (Segrest et al, 1971;Marciani and Papamatheakis, 1978). Additionally, we observed similar discrepancies with M r determination for P. alliacea alliinase, which shares with the P. alliacea LFS four of its five protein subunits (Musah et al, 2009). In-gel carbohydrate detection experiments showed that the a#-subunit is glycosylated (Fig.…”

“…Indeed, a single gene product can be a member of multiple complexes, such that the same protein or peptide can be utilized in different ways, depending upon the other peptides or proteins with which it is complexed. Surprisingly, even though the P. alliacea alliinase contains all the subunits of which the LFS is comprised, it is totally devoid of LFS activity (Musah et al, 2009). Likewise, the presence in the LFS of four of the five subunits contained in the P. alliacea alliinase does not render the LFS able to catalyze the breakdown of S-substituted Cys sulfoxides.…”

“…After comparison of the SDS-PAGE results obtained for the LFS with those determined for the P. alliacea alliinase reported in the companion article (Musah et al, 2009), we were surprised to observe that the LFS appears to be identical to the alliinase, with the exception of the absence in the LFS of the b-subunit that is present in the alliinase (Fig. 5F).…”

“…1; Kubec et al, 2002) upon root tissue disruption. We have also shown that an alliinase that mediates the transformation of the petiveriins and 2-hydroxyethiins to their corresponding thiosulfinates is present in P. alliacea (Musah et al, 2009). Interestingly, while working with P. alliacea root extracts, we noted the presence of a potent lachrymator that we subsequently determined to be a sulfine-(Z)-phenylmethanethial S-oxide (PMTSO; Fig.…”

Two Novel Alliin Lyase (Alliinase) Genes from Twisted-Leaf Garlic (Allium obliquum) and Mountain Garlic (Allium senescens var. montanum)

“…The three-dimensional structure of the garlic alliinase at 1.5 Å resolution revealed that the active enzyme is a pyridoxal-5'-phosphate-dependent homodimeric glycoprotein (Kuettner et al, 2002), while another X-ray structure of the enzyme has improved the understanding of the pyridoxal phosphate binding to the active site (Shimon et al, 2007). A novel heteropentameric alliinase was detected and purified from the roots of the Amazonian medicinal plant Petiveria alliacea (Musah et al, 2009b), the same species displaying a novel lachrymatory factor synthase (LFS) (Musah et al, 2009a), an enzyme active only in the presence of alliinase (Eady et al, 2008). Another alliinase, purified from the bacterium Ensifer adaerens, displays a novel type of substrate specificity (Yutani et al, 2011).…”