RESUMEN
The extremely halophilic archaeon ZP-6 was isolated from Ai-Ding salt lake in Xinjiang Uighur Autonomous Region of the People's Republic of China. Based on its physiological properties, 16S rDNA sequence, and DNA-DNA homology with known haloarchaea, the isolate was tentatively identified as a Halobacterium sp. An acetoacetyl-CoA thiolase was purified and characterized from this organism. The native enzyme has a molecular mass of 80 +/- 8 kDa and consists of two identical subunits of 43 +/- 2 kDa each. The N-terminus 14 amino acid residues were sequenced and showed identity with the respective part of a putative thiolase (AcaB1) of Halobacterium sp. NRC-1. The purified enzyme has an optimal pH of 7.9 for acetoacetyl-CoA thiolysis. The thiolytic activity was inhibited by the presence of Mg'- and was stimulated by KCl or NaCl. The thiolysis reaction of Halobacterium sp. ZP-6 thiolase can be inhibited by either substrate when present in excess. The distinct kinetic profile indicates that the thiolase from Halobacterium sp. ZP-6 may have a different catalytic mechanism from the so-called ping-pong mechanism employed by other thiolases. To our knowledge, this is the first report of the purification and characterization of a halophilic thiolase from an archaeal species.
Asunto(s)
Acetil-CoA C-Acetiltransferasa/aislamiento & purificación , Halobacterium/enzimología , Halobacterium/aislamiento & purificación , Acetil-CoA C-Acetiltransferasa/química , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Acetiltransferasa/metabolismo , Secuencia de Aminoácidos , China , Halobacterium/genética , Concentración de Iones de Hidrógeno , Cinética , Magnesio/farmacología , Datos de Secuencia Molecular , Peso Molecular , Subunidades de Proteína , Homología de Secuencia de Aminoácido , Cloruro de Sodio/farmacología , Microbiología del AguaRESUMEN
5' and 3' end sequence of acaBl gene as primers, the gene of halophilic thiolase from haloarchae, Halobacterium sp. ZP-6 was cloned and its amino acid composition was calculated. Compared with non-halophilic thiolase, the halophilic thiolase contains more negative charge amino acid, less positive amino acid and less strong hydrophobic amino acid, and use preferably small side-chain amino acid. Those suggest that electrostatic screen, hydrophobic effect and surface tension all contribute to halophilic properties of thiolase.