RESUMEN
A 1.2 kb long DNA segment from Rhodospirillum rubrum has been sequenced (EMBL/GenBank accession number: U41280). This DNA segment includes the first sequenced gene for a putative 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, termed hmgL, from a photosynthetic organism. The sequenced segment also contains a ribosome-binding site and two clusters of possible-35 and -10 promotor sequences preceding the hmgL gene. Translation of the gene would yield a 303 amino-acid-long protein with a calculated molecular weight of 31.1 kDa. This protein shows 55-60% identity and approx. 75% similarity, including conservative substitutions, with the three eukaryotic and the single prokaryotic HMG-CoA lyases which previously have been sequenced. The R. rubrum enzyme showed stronger homology to the chicken HMG-CoA lyase than to the other bacterial protein. Significant sequence similarity was also found with homocitrate synthases from nitrogen-fixing prokaryotes. In contrast to the other sequenced prokaryotic HMG-CoA lyase (from Pseudomonas mevalonii), the R. rubrum hmgL does not seem to appear in an operon together with a HMG-CoA reductase. The hmgL gene was transcribed in photosynthetically grown cells as judged by amplification of cDNAs synthesised from DNA-free total RNA. In addition, HMG-CoA lyase activity was found in R. rubrum cells grown anaerobically in the light with leucine as the carbon source.
Asunto(s)
Genes Bacterianos , Oxo-Ácido-Liasas/genética , Rhodospirillum rubrum/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Operón , Oxo-Ácido-Liasas/clasificación , Oxo-Ácido-Liasas/metabolismo , Fotosíntesis , Filogenia , Regiones Promotoras Genéticas , ARN Bacteriano/genética , ARN Mensajero/genética , Rhodospirillum rubrum/enzimología , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Regiones Terminadoras Genéticas , Transcripción GenéticaRESUMEN
Comparison of the Arabidopsis thaliana vacuolar proton-pumping inorganic pyrophosphatase with three F0F1-ATPase c-subunits revealed a strong similarity between a stretch containing amino acids 227-245 of the H(+)-PPase and a transmembrane alpha-helix of the c-subunits which contains the glutamate which binds N,N'-dicyclohexylcarbodiimide.
Asunto(s)
Arabidopsis/enzimología , ATPasas de Translocación de Protón/química , Pirofosfatasas/química , Secuencia de Aminoácidos , Sitios de Unión , Diciclohexilcarbodiimida/metabolismo , Pirofosfatasa Inorgánica , Sustancias Macromoleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Rhodospirillum rubrum/enzimología , Homología de Secuencia de Aminoácido , Vacuolas/enzimologíaRESUMEN
Immunological cross-reactivity among three types of inorganic pyrophosphatases, that is, the proton pumping inorganic pyrophosphate synthase (H(+)-PPi synthase) and the soluble inorganic pyrophosphatase, both from Rhodospirillum rubrum, and the vacuolar membrane inorganic pyrophosphatase (H(+)-PPase) from mung bean (Vigna radiata), were examined by means of immunoblot analyses. Antibodies raised against the mung bean H(+)-PPase cross-reacted with the H(+)-PPi synthase from R. rubrum but not with the soluble PPase from R. rubrum. N,N'-dicyclohexylcarbodiimide (DCCD), which inhibits both synthesis and hydrolysis of PPi catalysed by purified and chromatophore H(+)-PPi synthase, binds to the enzyme as shown by fluorography of [14C]DCCD labelling. These results suggest that the R. rubrum H(+)-PPase share close structural similarities with the vacuolar H(+)-PPase from Mung bean.