Journal Article

Chlorophyllase as a Serine Hydrolase: Identification of a Putative Catalytic Triad

Tohru Tsuchiya, Takuo Suzuki, Takafumi Yamada, Hiroshi Shimada, Tatsuru Masuda, Hiroyuki Ohta and Ken-ichiro Takamiya

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 44, issue 1, pages 96-101
Published in print January 2003 | ISSN: 0032-0781
Published online January 2003 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcg011
Chlorophyllase as a Serine Hydrolase: Identification of a Putative Catalytic Triad

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Chlorophyllases (Chlases), cloned so far, contain a lipase motif with the active serine residue of the catalytic triad of triglyceride lipases. Inhibitors specific for the catalytic serine residue in serine hydrolases, which include lipases effectively inhibited the activity of the recombinant Chenopodium album Chlase (CaCLH). From this evidence we assumed that the catalytic mechanism of hydrolysis by Chlase might be similar to those of serine hydrolases that have a catalytic triad composed of serine, histidine and aspartic acid in their active site. Thus, we introduced mutations into the putative catalytic residue (Ser162) and conserved amino acid residues (histidine, aspartic acid and cysteine) to generate recombinant CaCLH mutants. The three amino acid residues (Ser162, Asp191 and His262) essential for Chlase activity were identified. These results indicate that Chlase is a serine hydrolase and, by analogy with a plausible catalytic mechanism of serine hydrolases, we proposed a mechanism for hydrolysis catalyzed by Chlase.

Keywords: Keywords: Catalytic triad — Chenopodium album — Chlorophyllase (EC 3.1.1.14) — Chlorophyll degradation — Serine hydrolase.; Abbreviations: Chlase, Chlorophyllase; Chlide, chlorophyllide; DFP, diisopropyl fluorophosphate; EST, expressed sequence tag; NEM, N-ethylmaleimide; PCMB, p-chloromercuribenzoic acid; PMSF, phenylmethanesulfonyl fluoride.

Journal Article.  2981 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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