Rubredoxins Involved in Alkane Oxidation

Abstract

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.Rubredoxins (Rds) are the simplest of the iron-sulfur redox active proteins and usually contain a single Fe(S-Cys) 4 site. In the aerobic bacterium Pseudomonas putida GPo1, Rd is an essential component of the alkane hydroxylase system. It shuttles electrons from Rd reductase, a protein which reduces its flavin adenine dinucleotide at the expense of NADH, to alkane hydroxylase (19,21,26,28,39). The alkane hydroxylase belongs to a large class of integral membrane proteins (9, 42) that contain an oxobridged diiron cluster (32) and is of interest as a biocatalyst for the production of alcohols, fatty acids, and epoxides (24,44).Sequencing of the GPo1 alk genes, reviewed in reference 43, showed that the OCT plasmid of GPo1 encodes two Rds, AlkF and AlkG (14), the second of which has been studied in detail in earlier biochemical studies (see below). AlkG is unusual in that it is more than three times the size of other bacterial Rds. It consists of two Rd domains, AlkG1 and AlkG2, connected by a 70-amino-acid linker which shows no homology to other sequences in the Swiss-Prot database (3, 14, 27). AlkF has not been isolated or detected. The primary sequence of AlkF consists of an Rd-like N-terminal domain (AlkF1) and an 80-amino-acid C-terminal extension. AlkF1 and the N-terminal domain of AlkG (AlkG1) are more closely related to each other than to the C-terminal domain of AlkG (AlkG2) (see Fig. 3). Only plasmids which encode AlkG2 were able to restore growth on n-alkanes in P. putida carrying a CAM-OCT alkA mutant plasmid (14). Two Rd genes cloned ...

Crystal structure of the electron transfer complex rubredoxin–rubredoxin reductase of Pseudomonas aeruginosa

“…Sequence alignments indicate that Rdxs involved in alkane oxidation fall into two classes, denoted AlkG1-and AlkG2-type Rdxs (9). RubA2 and RubA1 of P. aeruginosa PAO1 are both AlkG2-type Rdxs, are encoded by neighboring genes, and are 80% identical by amino acid sequence.…”

“…Only the latter transfers electrons from RdxR to alkane hydroxylases (9). In AlkG1-type Rdxs, an additional arginine is inserted immediately downstream of the second metal-binding CXXCG motif.…”

“…The membrane-bound alkane hydroxylases AlkB1 and AlkB2 oxidize terminal carbons of chemically inert n-alkanes, allowing further oxidation and degradation. Electrons for this initial reaction derive from carbon metabolism relayed through an electron transport (ET) chain involving FAD-dependent NAD(P)H:rubredoxin reductase (RdxR) RubB and two AlkG2-type (see below) rubredoxins (Rdxs) RubA2 and RubA1 (7)(8)(9), encoded by the gene cluster rubB (PA5349), rubA2 (PA5350), and rubA1 (PA5351). Whereas alkB1 and alkB2 expression is strictly n-alkane-dependent, RubB/RubA1/RubA2 are constitutively produced (8), indicating a more general but as yet unidentified role for this ET chain in P. aeruginosa (7).…”

Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene

“…Several rubredoxins from alkane-degrading bacteria have been cloned and analysed in complementation assays for their ability to substitute P. putida GPo1 AlkG. They clustered into two groups: AlkG1-type rubredoxins cannot transfer electrons to the alkane hydroxylase in GPo1, while AlkG2-type enzymes can do so and can substitute for AlkG [169]. AlkG1-type rubredoxins probably have…”

Quantitative PCR analysis of diesel degrading genes of Acinetobacter calcoaceticus isolates

“…Previous studies showed that Acinetobacter sp. strain ADP1 requires at least five essential genes, namely rubA, rubB, alkM, alkR, and xcpR, for n-alkane utilisation (Ratajczak et al, 1998) confirming rubA, rubB, and alkM constitute a three component alkane hydroxyase system in A. calcoaceticus (Ratajczak et al, 1998;Geißdörfer et al, 1999;van Beilen et al, 2002). The overall alkR gene expression of LT 1 , samples was comparatively low in relation to the alkane hydroxylase complex genes, alkM and rubA.…”