Purification and characterisation of the BIOH protein from the biotin biosynthetic pathway.
Conversion of pimeloyl-coenzyme A (CoA) to biotin in Escherichia coli requires at least four enzymes encoded by genes in the bio operon. One gene, bioH, which is not present in the bioABFCD operon, is required for the synthesis of pimeloyl-CoA but its exact role in formation of this intermediate is unknown. To investigate this further, we have overexpressed and purified the bioH gene products from both E. coli (BIOH EC) and Neisseria meningitis (BIOH NM) in E. coli. When purified BIOH was incubated with excess CoA and analysed by electrospray mass spectrometry a species of mass corresponding to a BIOH:CoA complex was observed. Mutation of a conserved serine residue to alanine (BIOH EC S82A) did not prevent CoA binding. This is the first report of the purification of BIOH and the observation of a small molecule bound to the protein provides clues to its role in pimeloyl-CoA synthesis.
Tomczyk
Nicholas H
NH
Department of Chemistry, Joseph Black Building, University of Edinburgh, West Mains Road, EH9 3JJ, Edinburgh, UK.
Nettleship
Joanne E
JE
Baxter
Robert L
RL
Crichton
Hilary J
HJ
Webster
Scott P
SP
Campopiano
Dominic J
DJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D001710:Biotin / Q000096:biosynthesis*; D003001:Cloning, Molecular; D003065:Coenzyme A / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D064429:Fatty Acid Synthases / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009343:Neisseria / Q000235:genetics / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013869:Thiolester Hydrolases / Q000737:chemistry
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C061417:bioH protein, E coli; D001710:Biotin; D064429:Fatty Acid Synthases; D013869:Thiolester Hydrolases; C022738:thioesterase II; D003065:Coenzyme A