title: Characterization of human β,β-carotene-15,15'-monooxygenase (BCMO1) as a soluble monomeric enzyme.
abstract: The formal first step in in vitamin A metabolism is the conversion of its natural precursor β,β-carotene (C40) to retinaldehyde (C20). This reaction is catalyzed by the enzyme β,β-carotene-15,15'-monooxygenase (BCMO1). BCMO1 has been cloned from several vertebrate species, including humans. However, knowledge about this protein's enzymatic and structural properties is scant. Here we expressed human BCMO1 in Spodoptera frugiperda 9 insect cells. Recombinant BCMO1 is a soluble protein that displayed Michaelis-Menten kinetics with a KM of 14 μM for β,β-carotene. Though addition of detergents failed to increase BCMO1 enzymatic activity, short chain aliphatic detergents such as C8E4 and C8E6 decreased enzymatic activity probably by interacting with the substrate binding site. Thus we purified BCMO1 in the absence of detergent. Purified BCMO1 was a monomeric enzymatically active soluble protein that did not require cofactors and displayed a turnover rate of about 8 molecules of β,β-carotene per second. The aqueous solubility of BCMO1 was confirmed in mouse liver and mammalian cells. Establishment of a protocol that yields highly active homogenous BCMO1 is an important step towards clarifying the lipophilic substrate interaction, reaction mechanism and structure of this vitamin A forming enzyme.
authors.affiliation: Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kowatz
authors.forename: Darwin
authors.initials: D
authors.lastname: Babino
authors.forename: Philip
authors.initials: P
authors.lastname: Kiser
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Palczewski
authors.forename: Johannes
authors.initials: J
authors.lastname: von Lintig
mesh_terms: D000818:Animals; D002460:Cell Line; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008099:Liver / Q000201:enzymology; D051379:Mice; D011994:Recombinant Proteins / Q000302:isolation & purification; D012172:Retinaldehyde / Q000737:chemistry; D012995:Solubility; D018411:Spodoptera / Q000201:enzymology; D013379:Substrate Specificity / Q000235:genetics; D014162:Transfection; D014801:Vitamin A / Q000737:chemistry / Q000378:metabolism; D050559:beta-Carotene 15,15'-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D014801:Vitamin A; C493358:BCO1 protein, human; D050559:beta-Carotene 15,15'-Monooxygenase; D012172:Retinaldehyde
keywords: All-trans-retinal; Cytosolic; Non-heme iron oxygenase; Symmetric carotenoid cleavage; Vitamin A; β,β-Carotene; β,β-Carotene-15,15′-monooxygenase