A light-dependent complementation system for analysis of NADPH:protochlorophyllide oxidoreductase: Identification and mutagenesis of two conserved residues that are essential for enzyme activity
- Univ. of Virginia, Charlottesville, VA (United States)
Protochlorophyllide reductase (NADPH:protochlorophyllide oxidoreductase; EC 1.6.99.1) catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory step in the chlorophyll biosynthetic pathway. We have developed an expression system in which the protochlorophyllide reductase from pea (Pisum sativum L.) is used to complement protochlorophyllide reduction mutants in the photosynthetic bacterium Rhodobacter capsulatus, allowing analysis of wild-type and mutant forms of the enzyme. By protein sequence comparisons, we have identified the plant protochlorophyllide reductases as belonging to the family of short-chain alcohol dehydrogenases. Based on our protein sequence alignments, we have identified and mutated two conserved residues (Tyr-275 and Lys-279) within the proposed active site of the enzyme and shown that they are critical for activity. A model of the enzyme reaction mechanism for light-dependent protochlorophyllide reduction is proposed. 33 refs., 5 figs.
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- FG05-94ER20144
- OSTI ID:
- 50802
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 3 Vol. 92; ISSN PNASA6; ISSN 0027-8424
- Country of Publication:
- United States
- Language:
- English
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