The green odor of plants is characterized by green leaf volatiles (GLVs) composed of C6 compounds. GLVs are biosynthesized from polyunsaturated fatty acids in thylakoid membranes by a series of enzymes. A representative member of GLVs (E)-2-hexenal, known as the leaf aldehyde, has been assumed to be produced by isomerization from (Z)-3-hexenal in the biosynthesis pathway; however, the enzyme has not yet been identified. In this study, we purified the (Z)-3:(E)-2-hexenal isomerase (HI) from paprika fruits and showed that various plant species have homologous HIs. Purified HI is a homotrimeric protein of 110 kDa composed of 35-kDa subunits and shows high activity at acidic and neutral pH values. Phylogenetic analysis showed that HIs belong to the cupin superfamily, and at least three catalytic amino acids (His, Lys, Tyr) are conserved in HIs of various plant species. Enzymatic isomerization of (Z)-3-hexenal in the presence of deuterium oxide resulted in the introduction of deuterium at the C4 position of (E)-2-hexenal, and a suicide substrate 3-hexyn-1-al inhibited HI irreversibly, suggesting that the catalytic mode of HI is a keto-enol tautomerism reaction mode mediated by a catalytic His residue. The gene expression of HIs in Solanaceae plants was enhanced in specific developmental stages and by wounding treatment. Transgenic tomato plants overexpressing paprika HI accumulated (E)-2-hexenal in contrast to wild-type tomato plants mainly accumulating (Z)-3-hexenal, suggesting that HI plays a key role in the production of (E)-2-hexenal in planta.
Symplectin is a photoprotein from a luminous squid, Symplectoteuthis oualaniensis. It has a luminous substrate, dehydrocoelenterazine (DCZ), linked through a thioether bond with a cysteine residue. We have proven the binding site of luminous substrate in symplectin by using an artificial analogue of DCZ, ortho-fluoro-DCZ (F-DCZ). F-DCZsymplectin emitting strong blue light was reconstituted from apo-symplectin and F-DCZ. Proteolytic digestion of the reconstituted F-DCZ-symplectin afforded peptides including C 390 GLK-F-DCZ (amide), which was detected with a house assembled nano-LC-ESI-Q-TOF-MS. The chromo-peptide derived from the F-DCZ-symplectin after luminescence showed the lower molecular mass than that before the luminescence by 12 mass units, corresponding to the loss of one carbon atom upon emitting light. Thus, we have concluded that F-DCZ analogue binds to Cys390 in symplectin so as to emit light.
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