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ARS Home » Midwest Area » Columbia, Missouri » Plant Genetics Research » Research » Publications at this Location » Publication #170865
Title: MECHANISTIC ANALYSIS OF WHEAT CHLOROPHYLLASE

Author
item ARKUS, KIANI - DANFORTH PLANT SCIENCE CT
item Cahoon, Edgar
item JEZ, JOSEPH - DANFORTH PLANT SCIENCE CT

Submitted to: Archives of Biochemistry and Biophysics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/25/2005
Publication Date: 6/1/2005
Citation: Arkus, K., Cahoon, E.B., Jez, J. 2005. Mechanistic analysis of wheat chlorophyllase. Archives Of Biochemistry and Biophysics. 438:146-155.

Interpretive Summary: Chlorophyllase is an enzyme that catalyzes the first step in chlorophyll breakdown. The ability to regulate the activity of this enzyme has important implications for the production of horticultural and agronomic crops, such as delaying the senescence of cut flowers and vegetables and removing contaminating chlorophyll from vegetable oils. Chlorophyllase also has the potential for use in detergent formulations to remove grass stains. The kinetic properties of chlorophyllase from wheat are described in this paper. This enzyme is shown to have activity over a wide range of temperatures, including temperatures as high as 167°F. The wheat chlorophyllase is further shown to function on alternative substrates that have chemical structures slightly different than that of chlorophyll. Information in this paper will be useful for biochemists and geneticists attempting to modify the activity of chlorophyllase for improved horticultural and agronomic products as well as for industrial chemists attempting to devise new detergents. This work may ultimately lead to agricultural products with improved properties, including extended shelf-life, and detergents with improved ability to remove grass stains.

Technical Abstract: Chlorophyllase (chlorophyll-chlorophyllidohydrolase, EC 3.1.1.14) catalyzes the hydrolysis of chlorophyll into chlorophyllide and phytol as the first step in degradation of the pigment. The biological function of the enzyme has industrial and agricultural applicability as a biocatalyst. Although chlorophyllase activity in plants is widespread, protein aggregation during extraction from plant tissues and the limited yields obtained from chloroplasts have limited efforts to examine its reaction mechanism. Here we report the cloning of chlorophyllase from Triticum aestivum (wheat), which shares ~40% amino acid identity with the chlorophyllases from Chenopodium, Arabidopsis, and Citrus, and provide the first detailed kinetic analysis of a purified chlorophyllase. Purification of recombinant protein from a heterologous E. coli expression system indicates that chlorophyllase functions as a dimeric protein. Wheat chlorophyllase hydrolyzed the phytol moiety from chlorophyll with kcat = 566 min-1 and Km = 63 uM. The enzyme was also active over a broad temperature range (10 - 75 °C). Our kinetic analysis of wheat chlorophyllase supports the hypothesis that the enzyme uses a charge-relay mechanism similar to other carboxylesterases for catalysis.