Abstract
Serpin protease inhibitors and β-amylase starch hydrolases are very abundant seed proteins in the endosperm of grasses. β-amylase is a crucial enzyme in the beer industry providing maltose for fermenting yeast. In animals and plants, inhibitory serpins form covalent linkages that inactivate their cognate proteases. Additionally, in animals, noninhibitory functions for serpins are observed such as metabolite carriers and chaperones. The function of serpins in seeds has yet to be unveiled. In developing endosperm, serpin Z4 and β-amylase showed similar in vivo spatio-temporal accumulation properties and colocalize in the cytosol of transformed tobacco leaves. A molecular interaction between recombinant proteins of serpin Z4 and β-amylase was revealed by surface plasmon resonance and microscale thermophoresis yielding a dissociation constant of 10-7 M. Importantly, the addition of serpin Z4 significantly changes β-amylase enzymatic properties by increasing its maximal catalytic velocity. The presence of serpin Z4 stabilizes β-amylase activity during heat treatment without affecting its critical denaturing temperature. Oxidative stress, simulated by the addition of CuCl2, leads to the formation of high molecular weight polymers of β-amylase similar to those detected in vivo. The polymers were cross-linked through disulfide bonds, the formation of which was repressed when serpin Z4 was present. The results suggest an unprecedented function for a plant seed serpin as a β-amylase-specific chaperone-like partner that could optimize β-amylase activity upon germination. This report is the first to describe a noninhibitory function for a serpin in plants.