RESUMO
Two pathways for cysteine biosynthesis are known in nature; however, it is not known which, if either, the Archaea utilize. Enzyme activities in extracts of Methanosarcina thermophila grown with combinations of cysteine and sulfide as sulfur sources indicated that this archaeon utilizes the pathway found in the Bacteria domain. The genes encoding serine transacetylase and O-acetylserine sulfhydrylase (cysE and cysK) are adjacent on the chromosome of M. thermophila and possibly form an operon. When M. thermophila is grown with cysteine as the sole sulfur source, O-acetylserine sulfhydrylase activity is maximally expressed suggesting alternative roles for this enzyme apart from cysteine biosynthesis.
Assuntos
Carbono-Oxigênio Liases/metabolismo , Cisteína/biossíntese , Methanosarcina/metabolismo , Complexos Multienzimáticos , Proteínas de Saccharomyces cerevisiae , Enxofre/metabolismo , Sequência de Aminoácidos , Carbono-Oxigênio Liases/genética , Cisteína Sintase , Methanosarcina/genética , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Cysteine is the major source of fixed sulfur for the synthesis of sulfur-containing compounds in organisms of the Bacteria and Eucarya domains. Though pathways for cysteine biosynthesis have been established for both of these domains, it is unknown how the Archaea fix sulfur or synthesize cysteine. None of the four archaeal genomes sequenced to date contain open reading frames with identities to either O-acetyl-L-serine sulfhydrylase (OASS) or homocysteine synthase, the only sulfur-fixing enzymes known in nature. We report the purification and characterization of OASS from acetate-grown Methanosarcina thermophila, a moderately thermophilic methanoarchaeon. The purified OASS contained pyridoxal 5'-phosphate and catalyzed the formation of L-cysteine and acetate from O-acetyl-L-serine and sulfide. The N-terminal amino acid sequence has high sequence similarity with other known OASS enzymes from the Eucarya and Bacteria domains. The purified OASS had a specific activity of 129 micromol of cysteine/min/mg, with a K(m) of 500 +/- 80 microM for sulfide, and exhibited positive cooperativity and substrate inhibition with O-acetyl-L-serine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band at 36 kDa, and native gel filtration chromatography indicated a molecular mass of 93 kDa, suggesting that the purified OASS is either a homodimer or a homotrimer. The optimum temperature for activity was between 40 and 60 degrees C, consistent with the optimum growth temperature for M. thermophila. The results of this study provide the first evidence for a sulfur-fixing enzyme in the Archaea domain. The results also provide the first biochemical evidence for an enzyme with the potential for involvement in cysteine biosynthesis in the Archaea.
Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Carbono-Oxigênio Liases/química , Carbono-Oxigênio Liases/metabolismo , Methanosarcina/enzimologia , Complexos Multienzimáticos , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Proteínas Arqueais/isolamento & purificação , Carbono-Oxigênio Liases/isolamento & purificação , Cisteína/biossíntese , Cisteína Sintase , Dimerização , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Cinética , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Serina/análogos & derivados , Serina/química , Serina/metabolismo , Espectrofotometria Ultravioleta , Sulfetos/química , Sulfetos/metabolismo , Enxofre/metabolismoRESUMO
Organochlorine pesticide levels were determined in Neotropical migrant passerines which have populations reported to be declining. Pesticide residues ranging from 0.385 to 27.4 ng/g were found in 19 of 21 birds, including both Hatch Year (HY) and After Hatch Year (AHY) age classes of nine different species. Eighteen of the 19 birds contained p,p'-DDE, while dieldrin was found in 16 birds and heptachlor epoxide was present in 10 birds. There were no significant differences in pesticide levels between HY and AHY birds, suggesting that HY birds are exposed to pesticides in their diet on the breeding ground or that pesticides are acquired from contaminated mothers, or both. Pesticide levels were not related to gender. Although the effects of these low residue levels on passerine fecundity are unknown, their presence has potential conservation implications for passerines and for the raptorial birds that feed upon them.