Purification and characterization of an extremely halophilic acetoacetyl-CoA thiolase from a newly isolated Halobacterium strain ZP-6.

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.