Alkane Hydroxylase System Research Articles

Synthesis of alkane hydroxylase ofPseudomonas oleovorans increases the iron requirement ofalk+ bacterial strains

The alk genes enable Pseudomonas oleovorans to utilize alkanes as sole carbon and energy source. Expression of the alk genes in P. oleovorans and in two Escherichia coli recombinants induced iron limitation in minimal medium cultures. Further investigation showed that the expression of the alkB gene, encoding the integral cytoplasmic membrane protein AlkB, was responsible for the increase of the iron requirement of E. coli W3110 (pGEc47). AlkB is the non-heme iron monooxygenase component of the alkane hydroxylase system, and can be synthesized to levels up to 10% (w/w) of total cell protein in E. coli W3110 (pGEc47). Its synthesis is, however, strictly dependent on the presence of sufficient iron in the medium. Our results show that a glucose-grown E. coli alk+ strain can reach alkane hydroxylase activities of about 25 U/g cdw, and are consistent with the recent finding that catalytically active AlkB contains two, rather than one iron atom per polypeptide chain.

Solubilization of the overexpressed integral membrane protein alkane monooxygenase of the recombinant Escherichia coli W3110[pGEc47

The integral membrane-bound alkane monooxygenase (AlkB) from Pseudomonas oleovorans has been overexpressed in the recombinant Escherichia coli strain W3110[pGEc47] and expression levels of 10 to 15% relative to the total cell protein were reached. The amount of phospholipids in induced cells is about 3-fold higher compared to the wild-type and AlkB has been shown to be located in small membrane vesicles. We present here a study on the solubilization of these AlkB containing membrane vesicles by different detergents with special emphasis on structural requirements for a surfactant preserving the activity of AlkB. Moreover, the effects of the detergents used on the complete alkane hydroxylase system was studied.

Substrate specificity of the alkane hydroxylase system of Pseudomonas oleovorans GPo1

We have studied the hydroxylation of a wide range of linear, branched, and cyclic alkanes and alkylbenzenes by the alkane hydroxylase system of Pseudomonas oleovorans GPo1 in vivo and in vitro. In vivo hydroxylation was determined with whole cells of the recombinant PpS8141; P. putida PpS81 carrying plasmid pGEc41. In vitro hydroxylation was determined with a reconstituted hydroxylase system consisting of AlkB (the membrane-bound catalytic monooxygenase component), AlkG (rubredoxin), and spinach ferredoxin reductase. The introduction of one or two methyl substituents in linear alkanes hampers, but does not block, the conversion of alkanes. However, substrates were not hydroxylated when a tertiary carbon was present. Substituted cyclic alkanes were oxidized with high enantiomeric excess at ring positions trans-4 relative to the substituents, but not at the methyl- or ethyl-substituents themselves. trans-1,4-dimethylcyclohexane and t-butylcyclohexane were not hydroxylated at all. Several alkylbenzenes, such as ethylbenzene and its 3- and 4-substituted derivatives, were hydroxylated at rates close to or superior to the rate for n-nonane. Isopropylbenzene and n-butylbenzene were oxidized at intermediate rates, while toluene and n-propylbenzene were converted at relatively low rates, only in vivo, and only when present in high concentrations. t-Butylbenzene was not oxidized at all. These results indicate that the P. oleovorans alkane hydroxylase system can be employed in the production of a considerable range of aliphatic and aromatic alcohols. The data also provide information on the dimensions of the substrate-binding site.

The alkane oxidation system of Pseudomonas oleovorans: induction of the alk genes in Escherichia coli W3110 (pGEc47) affects membrane biogenesis and results in overexpression of alkane hydroxylase in a distinct cytoplasmic membrane subfraction.

The alkane hydroxylase system of Pseudomonas oleovorans, which catalyses the initial oxidation of aliphatic substrates, is encoded by three genes. One of the gene products, the alkane hydroxylase AlkB, is an integral cytoplasmic membrane protein. Induction leads to the synthesis of 1.5-2% AlkB relative to the total cell protein, both in P. oleovorans and in recombinant Escherichia coli DH1. We present a study on the induction and localization of the alkane hydroxylase in E. coli W3110, which appears to be an interesting host strain because it permits expression levels of AlkB of up to 10-15% of the total cell protein. This expression level had negative effects on cell growth. The phospholipid content of such cells was about threefold higher than that of wild-type W3110. Freeze-fracture electron microscopy showed that induction of the alk genes led to the appearance of membrane vesicles in the cytoplasm; these occurred much more frequently in cells expressing alkB than in the negative control, which contained all of the alk genes except for alkB. Isolation and separation of the membranes of cells expressing alkB by density gradient centrifugation showed the customary cytoplasmic and outer membranes, as well as a low-density membrane fraction. This additional fraction was highly enriched in AlkB, as shown both by SDS-PAGE and enzyme activity measurements. A typical cytoplasmic membrane protein, NADH oxidase, was absent from the low-density membrane fraction. alkB expression in W3110 changed the composition of the phospholipid headgroup in the membrane, as well as the fatty acid composition of the membrane. The major changes occurred in the unsaturated fatty acids: C16:1 and C18:1 increased at the expense of C17:0cyc and C19:0cyc.

The induction of cytochrome P-450 in the alkane-utilizing yeast Lodderomyces elongisporus: Alterations in the microsomal membrane fraction

The adaptation of Lodderomyces elongisporus cells to n-alkane utilization was found to be connected with several alterations in the enzyme pattern of the whole cell and the microsomal fraction in particular. A strong induction was found for the microsomal localized cytochrome P-450 alkane hydroxylase system and other enzymes which are directly involved in the terminal degradation pathway of n-alkanes (long-chain alcohol and aldehyde dehydrogenases, catalase). The decrease of the pO2 in the medium enhances the concentration of the constituents of the alkane hydroxylase system as well as that of several other haemoproteins (catalase, cytochrome oxidase), while the long-chain alcohol and aldehyde dehydrogenase enzymes are probably unaffected.