title: Identification of an amino acid position that determines the substrate range of integral membrane alkane hydroxylases.
abstract: Selection experiments and protein engineering were used to identify an amino acid position in integral membrane alkane hydroxylases (AHs) that determines whether long-chain-length alkanes can be hydroxylated by these enzymes. First, substrate range mutants of the Pseudomonas putida GPo1 and Alcanivorax borkumensis AP1 medium-chain-length AHs were obtained by selection experiments with a specially constructed host. In all mutants able to oxidize alkanes longer than C13, W55 (in the case of P. putida AlkB) or W58 (in the case of A. borkumensis AlkB1) had changed to a much less bulky amino acid, usually serine or cysteine. The corresponding position in AHs from other bacteria that oxidize alkanes longer than C13 is occupied by a less bulky hydrophobic residue (A, V, L, or I). Site-directed mutagenesis of this position in the Mycobacterium tuberculosis H37Rv AH, which oxidizes C10 to C16 alkanes, to introduce more bulky amino acids changed the substrate range in the opposite direction; L69F and L69W mutants oxidized only C10 and C11 alkanes. Subsequent selection for growth on longer alkanes restored the leucine codon. A structure model of AHs based on these results is discussed.
authors.affiliation: ETH Zürich, Institute of Biotechnology, ETH-Hönggerberg HPT, Zürich, Switzerland. vanbeilen@biotech.biol.ethz.ch
authors.forename: Jan B
authors.initials: JB
authors.lastname: van Beilen
authors.forename: Theo H M
authors.initials: TH
authors.lastname: Smits
authors.forename: Franz F
authors.initials: FF
authors.lastname: Roos
authors.forename: Tobias
authors.initials: T
authors.lastname: Brunner
authors.forename: Stefanie B
authors.initials: SB
authors.lastname: Balada
authors.forename: Martina
authors.initials: M
authors.lastname: Röthlisberger
authors.forename: Bernard
authors.initials: B
authors.lastname: Witholt
mesh_terms: D000473:Alkanes / Q000378:metabolism; D000595:Amino Acid Sequence; D042926:Cytochrome P-450 CYP4A / Q000737:chemistry* / Q000378:metabolism; D044129:Halomonadaceae / Q000201:enzymology*; D006900:Hydroxylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016958:Pseudomonas putida / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000473:Alkanes; D042926:Cytochrome P-450 CYP4A