RESUMEN
Like mitochondria, hydrogenosomes compartmentalize crucial steps of eukaryotic energy metabolism; however, this compartmentalization differs substantially between mitochondriate aerobes and hydrogenosome-containing anaerobes. Because hydrogenosomes have arisen independently in different lineages of eukaryotic microorganisms, comparative analysis of the various types of hydrogenosomes can provide insights into the functional and evolutionary aspects of compartmentalized energy metabolism in unicellular eukaryotes.
Asunto(s)
Metabolismo Energético , Células Eucariotas/metabolismo , Hidrógeno/metabolismo , Orgánulos/genética , Orgánulos/metabolismo , Adaptación Fisiológica , Aerobiosis , Anaerobiosis , Compartimento Celular , Células Eucariotas/ultraestructura , MitocondriasRESUMEN
The chytrid fungi Piromyces sp. E2 and Neocallimastix sp. L2 are obligatory amitochondriate anaerobes that possess hydrogenosomes. Hydrogenosomes are highly specialized organelles engaged in anaerobic carbon metabolism; they generate molecular hydrogen and ATP. Here, we show for the first time that chytrid hydrogenosomes use pyruvate formate-lyase (PFL) and not pyruvate:ferredoxin oxidoreductase (PFO) for pyruvate catabolism, unlike all other hydrogenosomes studied to date. Chytrid PFLs are encoded by a multigene family and are abundantly expressed in Piromyces sp. E2 and Neocallimastix sp. L2. Western blotting after cellular fractionation, proteinase K protection assays and determinations of enzyme activities reveal that PFL is present in the hydrogenosomes of Piromyces sp. E2. The main route of the hydrogenosomal carbon metabolism involves PFL; the formation of equimolar amounts of formate and acetate by isolated hydrogenosomes excludes a significant contribution by PFO. Our data support the assumption that chytrid hydrogenosomes are unique and argue for a polyphyletic origin of these organelles.
Asunto(s)
Acetiltransferasas/metabolismo , Hidrógeno/metabolismo , Orgánulos/enzimología , Piromyces/enzimología , Ácido Pirúvico/metabolismo , Acetiltransferasas/química , Acetiltransferasas/genética , Secuencia de Aminoácidos , Anaerobiosis , Secuencia de Bases , Northern Blotting , Western Blotting , ADN Complementario , ADN de Hongos , Endopeptidasa K/metabolismo , Biblioteca de Genes , Genes Fúngicos , Mitocondrias/enzimología , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Piromyces/genética , Alineación de SecuenciaRESUMEN
The anaerobic chytrid Piromyces sp. E2 lacks mitochondria, but contains hydrogen-producing organelles, the hydrogenosomes. We are interested in how the adaptation to anaerobiosis influenced enzyme compartmentalization in this organism. Random sequencing of a cDNA library from Piromyces sp. E2 resulted in the isolation of cDNAs encoding malate dehydrogenase, aconitase and acetohydroxyacid reductoisomerase. Phylogenetic analysis of the deduced amino acid sequences revealed that they are closely related to their mitochondrial homologues from aerobic eukaryotes. However, the deduced sequences lack N-terminal extensions, which function as mitochondrial leader sequences in the corresponding mitochondrial enzymes from aerobic eukaryotes. Subcellular fractionation and enzyme assays confirmed that the corresponding enzymes are located in the cytosol. As anaerobic chytrids evolved from aerobic, mitochondria-bearing ancestors, we suggest that, in the course of the adaptation from an aerobic to an anaerobic lifestyle, mitochondrial enzymes were retargeted to the cytosol with the concomitant loss of their N-terminal leader sequences.
Asunto(s)
Aconitato Hidratasa/clasificación , Oxidorreductasas de Alcohol/clasificación , Malato Deshidrogenasa/clasificación , Piromyces/enzimología , Regiones no Traducidas 5' , Aconitato Hidratasa/genética , Aconitato Hidratasa/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Anaerobiosis , Secuencia de Bases , Citosol/enzimología , ADN Complementario , ADN de Hongos , Dosificación de Gen , Expresión Génica , Cetoácido Reductoisomerasa , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Mitocondrias/enzimología , Datos de Secuencia Molecular , Piromyces/genética , ARN de Hongos , ARN MensajeroRESUMEN
Cofactor extracts of Methanogenium tationis were screened for the presence of pterin-derivatives. Methanopterin, sarcinapterin and 7-methylpterin were absent, while 2-amino-4-hydroxy-pteridine and another blue fluorescent compound with a pterin spectrum were detected. The latter pterin was purified by ion exchange and reversed-phase column chromatography. The structure of this compound was elucidated by combining spectrophotometry, amino acid analysis and 1H-NMR spectroscopy. The pterin, which we named tatiopterin, was identified as an aspartyl derivative of sarcinapterin with a 7-proton instead of a 7-methyl group in the pterin moiety. The IUPAC name is: N-[-1'-(2''-amino-4''-hydroxy-7''-proton-6''-pteridinyl)ethyl]-4- [2',3',4',5'-tetrahydroxypent-1'-yl(5'----1'')O-alpha- ribofuranosyl-5''-phosphoric acid]aniline, in which the phosphate group is esterified with alpha-hydroxyglutarylglutamylaspartic acid.