Alkane Hydroxylase

This enzyme is three component monooxygenase, comprising a hydroxylase, a rubredoxin and rubredoxin reductase (Shanklin et al. 1997). The hydroxylase component is membrane-bound, while both rubredoxin and rubredoxin reductase components are soluble and cytoplasmic proteins. This enzymatic complex is able to oxidize medium and long chain linear alkanes using reducing equivalents from NADH or NADPH.

AlkB, an integral membrane protein, carries out a terminal hydroxylation of n-alkane (Kok et al. 1989). The electrons needed to carry out this step are delivered to AlkB via a rubredoxin reductase (AlkT) and two rubredoxins (AlkF and AlkG) (van Beilen et al. 2002). The resulting alcohol is further converted to a fatty acid via a pathway involving an alcohol dehydrogenase (AlkJ), an aldehyde dehydrogenase (AlkH) and an acyl-CoA synthetase (AlkK), that enters the b oxidation pathway (van Beilen et al. 2001). The histidine residues are required for activity in the members of this family (Shanklin et al. 1994). There is a conserved NYXEHYG(L/M) motif in all identified alkane hydroxylases (Smits et al. 2002). This motif has been proposed as a signature for membrane-bound alkane hydroxylases (Smits et al. 2002).

Although crystal structure of Alk is not known, it is believed to have six transmembrane segments and a catalytic site that faces the cytoplasm. The active site includes four His-containing sequence motives that are conserved in other hydrocarbon monooxygenases which chelate two iron atoms (Shanklin et al. 1994). The diiron cluster allows the O2-dependent activation of the alkane through a substrate radical intermediate (Shanklin et al. 1997; Bertrand et al. 2005). One of the O2 atoms is transferred to the terminal methyl group of the alkane, rendering an alcohol, while the other one is reduced to H2O by electrons transferred by the rubredoxin. Oxidation is regio- and stereospecific (van Beilen et al. 1995).

Baptist et al. (1963) have identified an enzyme system from Pseudomonas putida PpG6 grown on alkanes which is capable of oxidizing octane to octanoic acid, and the properties of the enzyme complex, which catalyzes the initial hydroxylation reaction, have been extensively studied (Mckenna and Coon 1970). In vitro, this hydroxylase complex is also capable of omega-oxidizing fatty acids (Mckenna and Coon 1970). This suggests that the oxidation of alkane and fatty acid chains might occur from both ends in strains with a functional hydroxylase.

The AlkB protein from Pseudomonas putida GPo1 is presently the best characterized Alk (van Beilen et al. 1994). It catalyses the first step of alkane degradation with the help of two electron transfer proteins, rubredoxin (AlkG) and rubredoxin reductase (AlkT) (van Beilen et al. 1994). Over the past decade, alkB-like hydrox-ylase genes have been detected in a wide range of alkane degrading bacteria, including a-, b- and g-proteobacteria; as well as in some high G + C content Grampositive bacteria (Smits et al. 2002). Many of these contain more than one alkB homologue, such as Pseudomonas aeruginosa PAO1 (alkB1 and alkB2), Rhodococcus erythropolis Q15 (alkB1-4) and Acinetobacter sp. M1 (alkMa and alkMb).

The enzymes, that oxidize alkanes larger than C20, seem to be totally different. For example, Acinetobacter sp. M1, which can grow on C13-C44 alkanes, contains a soluble, Cu2+ -dependent Alk that is active on C10-C30 alkanes. It has been proposed to be a dioxygenase that generates n-alkyl hydroperoxides to render the corresponding aldehydes (Tani et al. 2001). A different Acinetobacter strain, DSM 17874, has been found to contain a flavin-binding monooxygenase, named AlmA, which oxidizes C20 to >C32 alkanes (Throne-Holst et al. 2007). Genes homologous to almA have been identified in several other long chain n-alkane degrading strains, including Acinetobacter sp. M1 and A. borkumensis SK2. A different long chain alkane hydroxylase, named LadA, has been characterized in Geobacillus thermodenitrificans NG80-2 (Feng et al. 2007). It oxidizes C15-C36 alkanes, generating primary alcohols. Its crystal structure has shown that it is a two-component flavin-dependent oxygenase belonging to the bacterial luciferase family of proteins (Li et al. 2008).

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