Pearl millet cysteine protease inhibitor. Evidence for the presence of two distinct sites responsible for anti-fungal and anti-feedent activities.

Recently, pearl millet cysteine protease inhibitor (CPI) was, for the first time, shown to possess anti-fungal activity in addition to its anti-feedent (protease inhibitory) activity [Joshi, B.N. et al. (1998) Biochem. Biophys. Res. Commun. 246, 382-387]. Characterization of CPI revealed that it has a reversible mode of action for protease inhibition. The CD spectrum exhibited a 35% alpha helix and 65% random coil structure. The intrinsic fluorescence spectrum was typical of a protein devoid of tryptophan residues. Demetallation of Zn2+ resulted in a substantial change in the secondary and tertiary structure of CPI accompanied by the complete loss of anti-fungal and inhibitory activity indicating that Zn2+ plays an important role in maintaining both structural integrity and biological function. The differential response of anti-fungal and inhibitory activities to specific modifiers showed that there are two different reactive sites associated with anti-fungal and anti-feedent activity in CPI located on a single protein as revealed from its N-terminal sequence data (AGVCYGVLGNNLP). Modification of cysteine, glutamic/aspartic acid or argnine resulted in abolition of the anti-fungal activity of CPI, whereas modification of arginine led to an enhancement of the inhibitory activity in solution. Modification of histidine resulted in a twofold increase in the protease inhibitory activity without affecting the anti-fungal activity, whereas modification of serine led to selective inhibition of the protease inhibitory activity. The differential nature of the two activities was further supported by differences in the temperature stabilities of the anti-fungal (60 degrees C) and inhibitory (40 degrees C) activities. Binding of papain to CPI did not abolish the anti-fungal activity of CPI, supporting the presence of two active sites on CPI. The differential behavior of CPI towards anti-fungal and anti-feedent activity cannot be attributed to changes in conformation, as assessed by their CD and fluorescence spectra. The interaction of CPI modified for arginine or histidine with papain resulted in an enhancement of CPI activity accompanied by a slight decrease in fluorescence intensity of 15-20% at 343 nm. In contrast, modification of serine resulted in inhibition of CPI activity with a concomitant increase of 20% in the fluorescence intensity when complexed by the enzyme. This implies the involvement of enzyme-based tryptophan in the formation of a biologically active enzyme-inhibitor complex. The presence of anti-fungal and anti-feedent activity on a single protein, as evidenced in pearl millet CPI, opens up a new possibility of raising a transgenic plant resistant to pathogens, as well as pests, by transfer of a single CPI gene.

Cysteine protease inhibitor from pearl millet: a new class of antifungal protein.

A cysteine protease inhibitor exhibiting antifungal activity from pearl millet seeds has been purified to homogeneity by ammonium sulphate precipitation and chromatographic procedures involving CM- sephadex and SP-sepharose cation exchange columns. The molecular characterization has revealed its molecular mass as 24 kD and isoelectric point 9.8. The amino acid composition data shows presence of high content of serine and glycine (34 residues/mole) and absence of tryptophan. The inhibitor exhibits potent antifungal activity against Trichoderma reesei, a dead wood fungus with minimum inhibitory dose to inhibit mycelial growth or spore germination is as low as 1 microgram/ml (250 ng/disc). In addition to Trichoderma reesei, the antifungal activity is observed against some important phytopathogenic fungi, namely, Claviceps, Helminthosporium, Curvularia, Alternaria and Fusarium species. To the best of our knowledge, a cysteine protease inhibitor as an antifungal protein is reported for the first time from a plant system.