A light-dependent complementation system for analysis of NADPH:protochlorophyllide oxidoreductase: identification and mutagenesis of two conserved residues that are essential for enzyme activity.
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.
Wilks
H M
HM
Department of Biology, University of Virginia, Charlottesville 22903.
Timko
M P
MP
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics; D008027:Light; D008239:Lysine / Q000502:physiology*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D018532:Pisum sativum / Q000201:enzymology; D016354:Rhodobacter capsulatus / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D014443:Tyrosine / Q000502:physiology*
D014443:Tyrosine; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase; D008239:Lysine