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.
Smits
Theo H M
TH
Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Hönggerberg, CH-8093 Zürich, Switzerland.
Balada
Stefanie B
SB
Witholt
Bernard
B
van Beilen
Jan B
JB
D000473:Alkanes; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003470:Culture Media; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D006090:Gram-Negative Bacteria / Q000201:enzymology* / Q000235:genetics; D006094:Gram-Positive Bacteria / Q000201:enzymology* / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011551:Pseudomonas fluorescens / Q000201:enzymology / Q000235:genetics; D012416:Rubredoxins / Q000235:genetics; D017385:Sequence Homology; D013045:Species Specificity
D000473:Alkanes; D001426:Bacterial Proteins; D003470:Culture Media; D012416:Rubredoxins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A