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Journal of Bacteriology, January 2005, p. 85-91, Vol. 187, No. 1
0021-9193/05/$08.00+0     doi:10.1128/JB.187.1.85-91.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification of an Amino Acid Position That Determines the Substrate Range of Integral Membrane Alkane Hydroxylases

Jan B. van Beilen,^* Theo H. M. Smits, Franz F. Roos, Tobias Brunner, Stefanie B. Balada, Martina Röthlisberger, and Bernard Witholt

ETH Zürich, Institute of Biotechnology, ETH Hönggerberg HPT, Zürich, Switzerland

Received 29 July 2004/ Accepted 21 September 2004

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.

Present address: ENAC/ISTE/LBE, EPFL, Lausanne, Switzerland.

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