Document Detail

Biochemical characterization and FAD-binding analysis of oleate hydratase from Macrococcus caseolyticus.
MedLine Citation:
PMID:  22203098     Owner:  NLM     Status:  Publisher    
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.
Young-Chul Joo; Ki-Woong Jeong; Soo-Jin Yeom; Yeong-Su Kim; Yangmee Kim; Deok-Kun Oh
Related Documents :
17805838 - The structure of rat liver triglycerides.
24923648 - Lipid malnutrition and early development: a study of motor reflexes and electrocorticog...
16665128 - Amino acid composition along the transport pathway during grain filling in wheat.
15073018 - A novel method for estimating ancestral amino acid composition and its application to p...
6478828 - Leucine metabolism as a source of acetate in the synthesis of hyaluronic acid.
25434418 - Three new polyunsaturated lipids from a guangxi marine sponge haliclona sp.
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2011-12-19
Journal Detail:
Title:  Biochimie     Volume:  -     ISSN:  1638-6183     ISO Abbreviation:  -     Publication Date:  2011 Dec 
Date Detail:
Created Date:  2011-12-28     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  1264604     Medline TA:  Biochimie     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2011. Published by Elsevier Masson SAS.
Department of Bioscience and Biotechnology, Konkuk University, 1 Hayang-dong Gangjin-gu, Seoul 143-701, Republic of Korea.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Previous Document:  Ultrasonographic findings of Achilles tendon and plantar fascia in patients with calcium pyrophospha...
Next Document:  Polyol osmolytes stabilize native-like cooperative intermediate state of yeast hexokinase A at low p...