RESUMO
The purple photosynthetic bacterium Rhodobacter sphaeroides has within its genome a cluster of photosynthesis-related genes approximately 41 kb in length. In an attempt to identify genes involved in the terminal esterification stage of bacteriochlorophyll biosynthesis, a previously uncharacterized 5-kb region of this cluster was sequenced. Four open reading frames (ORFs) were identified, and each was analyzed by transposon mutagenesis. The product of one of these ORFs, bchG, shows close homologies with (bacterio)chlorophyll synthetases, and mutants in this gene were found to accumulate bacteriopheophorbide, the metal-free derivative of the bacteriochlorophyll precursor bacteriochlorophyllide, suggesting that bchG is responsible for the esterification of bacteriochlorophyllide with an alcohol moiety. This assignment of function to bchG was verified by the performance of assays demonstrating the ability of BchG protein, heterologously synthesized in Escherichia coli, to esterify bacteriochlorophyllide with geranylgeranyl pyrophosphate in vitro, thereby generating bacteriochlorophyll. This step is pivotal to the assembly of a functional photosystem in R. sphaeroides, a model organism for the study of structure-function relationships in photosynthesis. A second gene, orf177, is a member of a large family of isopentenyl diphosphate isomerases, while sequence homologies suggest that a third gene, orf427, may encode an assembly factor for photosynthetic complexes. The function of the remaining ORF, bchP, is the subject of a separate paper (H. Addlesee and C. N. Hunter, J. Bacteriol. 181:7248-7255, 1999). An operonal arrangement of the genes is proposed.
Assuntos
Carbono-Oxigênio Ligases/genética , Genes Bacterianos , Família Multigênica , Fotossíntese/genética , Rhodobacter sphaeroides/genética , Sequência de Aminoácidos , Bacterioclorofilas/metabolismo , Evolução Biológica , Sequência Conservada , Escherichia coli/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Insercional , Fases de Leitura Aberta , Oxirredutases/genética , Fosfatos de Poli-Isoprenil/metabolismo , Porfirinas/análise , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , Rhodobacter sphaeroides/enzimologia , Rhodobacter sphaeroides/crescimento & desenvolvimento , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Regiões Terminadoras GenéticasRESUMO
The bacteriochlorophyll of the purple photosynthetic bacterium Rhodobacter sphaeroides is esterified with phytol. The presence of this alcohol moiety is essential for the correct assembly of the photosynthetic apparatus. Despite this, and the fact that R. sphaeroides is widely used for the study of structure-function relationships in photosynthesis, the molecular genetics of the steps in which the alcohol is added and modified have not previously been investigated in this organism. Sequencing near the center of the photosynthesis gene cluster has now revealed the existence of an open reading frame encoding a putative 394-amino-acid polypeptide displaying strong homology with the products of a number of genes from other photosynthetic organisms, each proposed to be responsible for the reduction of the alcohol moiety of (bacterio)chlorophyll to phytol. An R. sphaeroides transposon mutant in this gene, bchP, possessed a structurally modified photosystem assembled with bacteriochlorophyll esterified with geranylgeraniol, rather than with phytol, implying that the product of this gene was geranylgeranyl-bacteriochlorophyll reductase. This identification was confirmed by the performance of in vitro assays using heterologously expressed protein, providing the first direct demonstration of the activity of a bchP gene product.
Assuntos
Genes Bacterianos , Oxirredutases/genética , Rhodobacter sphaeroides/enzimologia , Rhodobacter sphaeroides/genética , Sequência de Aminoácidos , Sequência de Bases , Carbono-Oxigênio Ligases , Cromatografia Líquida de Alta Pressão , Elementos de DNA Transponíveis , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Modelos Estruturais , Dados de Sequência Molecular , Família Multigênica , Mutagênese , Fases de Leitura Aberta , Mapeamento Físico do Cromossomo , Homologia de Sequência de AminoácidosRESUMO
A gene from the cyanobacterium Synechocystis sp. PCC 6803 has been cloned and sequenced, and subsequently used to partially complement a bchP mutant of the purple photosynthetic bacterium Rhodobacter sphaeroides. This mutant is blocked in the terminal hydrogenation steps of bchla biosynthesis and possesses only bchl esterified with geranylgeraniol. It also has a reduced cellular level of the light-harvesting LH2 complex, and the 850 nm absorbance maximum of LH2 is red-shifted by approximately 6 nm. Upon heterologous expression of the Synechocystis bchP homologue, not only are hydrogenated forms of bchlaGG detectable, but the level of LH2 is increased and the red-shift reversed by several nm. We conclude that this gene, which we term chlP, encodes the enzyme catalysing the stepwise hydrogenation of geranylgeraniol to phytol during chla biosynthesis.
