Affiliation:
1. ETH Zürich, Institute of Biotechnology, ETH Hönggerberg HPT, Zürich, Switzerland
Abstract
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 C
13
, 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 C
13
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 C
10
to C
16
alkanes, to introduce more bulky amino acids changed the substrate range in the opposite direction; L69F and L69W mutants oxidized only C
10
and C
11
alkanes. Subsequent selection for growth on longer alkanes restored the leucine codon. A structure model of AHs based on these results is discussed.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
105 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献