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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 GPol 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.sub.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.sub.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.sub.10] to [C.sub.16] alkanes, to introduce more bulky amino acids changed the substrate range in the opposite direction; L69F and L69W mutants oxidized only [C.sub.10] and [C.sub.11] alkanes. Subsequent selection for growth on longer alkanes restored the leucine codon. A structure model of AHs based on these results is discussed.

Pseudomonas sp. VLB120 uses styrene as a sole source of carbon and energy. The first step in this metabolic pathway is catalyzed by an oxygenase (StyA) and a NADH-flavin oxidoreductase (StyB). Both components have been isolated from wild-type Pseudomonas strain VLB120 as well as from recombinant Escherichia coli. StyA from both sources is a dimer, with a subunit size of 47 kDa, and catalyzes the enantioselective epoxidation of C[??]C double bonds. Styrene is exclusively converted to S-styrene oxide with a specific activity of 2.1 U [mg.sup.-1] ([k.sub.cat] = 1.6 [s.sup.-1]) and [K.sub.m] values for styrene of 0.45 [+ or -] 0.05 mM (wild type) and 0.38 [+ or -] 0.09 mM (recombinant). The epoxidation reaction depends on the presence of a NADH-flavin adenine dinucleotide (NADH-FAD) oxidoreductase for the supply of reduced FAD. StyB is a dimer with a molecular mass of 18 kDa and a NADH oxidation activity of 200 U [mg.sup.-1] ([k.sub.cat] [NADH] = 60 [s.sup.-1]). Steady-state kinetics determined for StyB indicate a mechanism of sequential binding of NADH and flavin to StyB. This enzyme reduces FAD as well as flavin mononucleotide and riboflavin. The NADH oxidation activity does not depend on the presence of StyA. During the epoxidation reaction, no formation of a complex of StyA and StyB has been observed, suggesting that electron transport between reductase and oxygenase occurs via a diffusing flavin.

Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences have been analyzed with attention to regulation and evolution of the alk genes. Only the alkB and alkS promoter regions are conserved, other than the coding regions of the GPo1 and P1 alk genes.

A study was conducted to show that most of the strains that possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases, using PCR. Result provided evidence that the newly cloned P450s are indeed alkane hydroxylases.

Studies the Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences. Metabolic pathway of alkane degradation; Role and cellular localization of Alk proteins; Regulation of the alk genes; Proteins encoded by the alk gene clusters of P. putida; DNA flanking the GPo1 and P1 alk gene clusters.