Dataset Information

Substrate specificity of purified recombinant human ?-carotene 15,15'-oxygenase (BCO1).

ABSTRACT: Humans cannot synthesize vitamin A and thus must obtain it from their diet. ?-Carotene 15,15'-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15-15' double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (kcat/Km). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of ?-carotene with a Vmax = 197.2 nmol retinal/mg BCO1 × h, Km = 17.2 ?M and catalytic efficiency kcat/Km = 6098 M(-1) min(-1). The enzyme also catalyzed the oxidative cleavage of ?-carotene, ?-cryptoxanthin, and ?-apo-8'-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of ?-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of ?-carotene. The shorter ?-apocarotenals (?-apo-10'-carotenal, ?-apo-12'-carotenal, ?-apo-14'-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-?-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.

SUBMITTER: dela Sena C

PROVIDER: S-EPMC3873565 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Substrate specificity of purified recombinant human β-carotene 15,15'-oxygenase (BCO1).

dela Seña Carlo C Narayanasamy Sureshbabu S Riedl Kenneth M KM Curley Robert W RW Schwartz Steven J SJ Harrison Earl H EH

The Journal of biological chemistry 20131101 52

Humans cannot synthesize vitamin A and thus must obtain it from their diet. β-Carotene 15,15'-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15-15' double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (kcat/Km). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-termina ...[more]

PMID: 24187135

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Project description:BackgroundAldehyde-deformylating oxygenase (ADO) is an important enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, ADO exhibits quite low chain-length specificity with respect to the substrates ranging from C4 to C18 aldehydes, which is not suitable for producing fuels with different properties or different chain lengths.ResultsBased on the crystal structures of cADOs (cyanobacterial ADO) with substrate analogs bound, some amino acids affecting the substrate specificity of cADO were identified, including the amino acids close to the aldehyde group and the hydrophobic tail of the substrate and those along the substrate channel. Using site-directed mutagenesis, selected amino acids were replaced with bulky ones introducing steric hindrance to the binding pocket via large functional groups. All mutants were overexpressed, purified and kinetically characterized. All mutants, except F87Y, displayed dramatically reduced activity towards C14,16,18 aldehydes. Notably, the substrate preferences of some mutants towards different chain-length substrates were enhanced: I24Y for n-heptanal, I27F for n-decanal and n-dodecanal, V28F for n-dodecanal, F87Y for n-decanal, C70F for n-hexanal, A118F for n-butanal, A121F for C4,6,7 aldehydes, V184F for n-dodecanal and n-decanal, M193Y for C6-10 aldehydes and L198F for C7-10 aldehydes. The impact of the engineered cADO mutants on the change of the hydrocarbon profile was demonstrated by co-expressing acyl-ACP thioesterase BTE, fadD and V184F in E. coli, showing that n-undecane was the main fatty alkane.ConclusionsSome amino acids, which can control the chain-length selectivity of substrates of cADO, were identified. The substrate specificities of cADO were successfully changed through structure-guided protein engineering, and some mutants displayed different chain-length preference. The in vivo experiments of V184F in genetically engineered E. coli proved the importance of engineered cADOs on the distribution of the fatty alkane profile. The results would be helpful for the production of fatty alk(a/e)nes in cyanobacteria with different properties.

Dataset Information

Substrate specificity of purified recombinant human ?-carotene 15,15'-oxygenase (BCO1).

Publications

Substrate specificity of purified recombinant human β-carotene 15,15'-oxygenase (BCO1).

Similar Datasets

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