Biochemical characterization and FAD-binding analysis of oleate hydratase from Macrococcus caseolyticus.
A putative fatty acid hydratase gene from Macrococcus caseolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was a 68 kDa dimer with a molecular mass of 136 kDa. The enzymatic products formed from fatty acid substrates by the putative enzyme were isolated with high purity (>99%) by solvent fractional crystallization at low temperature. After the identification by GC-MS, the purified hydroxy fatty acids were used as standards to quantitatively determine specific activities and kinetic parameters for fatty acids as substrates. Among the fatty acids evaluated, specific activity and catalytic efficiency (k(cat)/K(m)) were highest for oleic acid, indicating that the putative fatty acid hydratase was an oleate hydratase. Hydration occurred only for cis-9-double and cis-12-double bonds of unsaturated fatty acids without any trans-configurations. The maximum activity for oleate hydration was observed at pH 6.5 and 25 °C with 2% (v/v) ethanol and 0.2 mM FAD. Without FAD, all catalytic activity was abolished. Thus, the oleate hydratase is an FAD-dependent enzyme. The residues G29, G31, S34, E50, and E56, which are conserved in the FAD-binding motif of fatty acid hydratases (GXGXXG((A/S))X((15-21))E((D))), were selected by alignment, and the spectral properties and kinetic parameters of their alanine-substituted variants were analyzed. Among the five variants, G29A, G31A, and E56A showed no interaction with FAD and exhibited no activity. These results indicate that G29, G31, and E56 are essential for FAD-binding.
Department of Bioscience and Biotechnology, Konkuk University, 1 Hayang-dong Gangjin-gu, Seoul 143-701, Republic of Korea.
Young-Chul
YC
Joo
Ki-Woong
KW
Jeong
Soo-Jin
SJ
Yeom
Yeong-Su
YS
Kim
Yangmee
Y
Kim
Deok-Kun
DK
Oh
D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism*; D019301:Oleic Acid / Q000378:metabolism*; D011485:Protein Binding; D040141:Staphylococcaceae / Q000201:enzymology*
D005182:Flavin-Adenine Dinucleotide; D019301:Oleic Acid; D006836:Hydro-Lyases