Identification of an amino acid position that determines the substrate range of integral membrane alkane hydroxylases.

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.

Functional analysis of alkane hydroxylases from gram-negative and gram-positive bacteria.

We have cloned homologs of the Pseudomonas putida GPo1 alkane hydroxylase from Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens CHA0, Alcanivorax borkumensis AP1, Mycobacterium tuberculosis H37Rv, and Prauserella rugosa NRRL B-2295. Sequence comparisons show that the level of protein sequence identity between the homologs is as low as 35%, and that the Pseudomonas alkane hydroxylases are as distantly related to each other as to the remaining alkane hydroxylases. Based on the observation that rubredoxin, an electron transfer component of the GPo1 alkane hydroxylase system, can be replaced by rubredoxins from other alkane hydroxylase systems, we have developed three recombinant host strains for the functional analysis of the novel alkane hydroxylase genes. Two hosts, Escherichia coli GEc137 and P. putida GPo12, were equipped with pGEc47 Delta B, which encodes all proteins necessary for growth on medium-chain-length alkanes (C(6) to C(12)), except a functional alkane hydroxylase. The third host was an alkB knockout derivative of P. fluorescens CHA0, which is no longer able to grow on C(12) to C(16) alkanes. All alkane hydroxylase homologs, except the Acinetobacter sp. ADP1 AlkM, allowed at least one of the three hosts to grow on n-alkanes.

Rubredoxins involved in alkane oxidation.

Rubredoxins (Rds) are essential electron transfer components of bacterial membrane-bound alkane hydroxylase systems. Several Rd genes associated with alkane hydroxylase or Rd reductase genes were cloned from gram-positive and gram-negative organisms able to grow on n-alkanes (Alk-Rds). Complementation tests in an Escherichia coli recombinant containing all Pseudomonas putida GPo1 genes necessary for growth on alkanes except Rd 2 (AlkG) and sequence comparisons showed that the Alk-Rds can be divided in AlkG1- and AlkG2-type Rds. All alkane-degrading strains contain AlkG2-type Rds, which are able to replace the GPo1 Rd 2 in n-octane hydroxylation. Most strains also contain AlkG1-type Rds, which do not complement the deletion mutant but are highly conserved among gram-positive and gram-negative bacteria. Common to most Rds are the two iron-binding CXXCG motifs. All Alk-Rds possess four negatively charged residues that are not conserved in other Rds. The AlkG1-type Rds can be distinguished from the AlkG2-type Rds by the insertion of an arginine downstream of the second CXXCG motif. In addition, the glycines in the two CXXCG motifs are usually replaced by other amino acids. Mutagenesis of residues conserved in either the AlkG1- or the AlkG2-type Rds, but not between both types, shows that AlkG1 is unable to transfer electrons to the alkane hydroxylase mainly due to the insertion of the arginine, whereas the exchange of the glycines in the two CXXCG motifs only has a limited effect.