Rebecca E. Parales

Aromatic hydrocarbon dioxygenases in environmental biotechnology

Aromatic hydrocarbon dioxygenases belong to a large family of Rieske non-heme iron oxygenases. The dioxygenases have a broad substrate specificity and catalyze enantiospecific reactions with a wide range of substrates. These characteristics make them attractive synthons for the production of industrially and medically important chiral chemicals and also provide essential information for the development of bioremediation technology.

Structure of an aromatic-ring-hydroxylating dioxygenase-naphthalene 1,2-dioxygenase.

BACKGROUNDnPseudomonas sp. NCIB 9816-4 utilizes a multicomponent enzyme system to oxidize naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. The enzyme component catalyzing this reaction, naphthalene 1,2-dioxygenase (NDO), belongs to a family of aromatic-ring-hydroxylating dioxygenases that oxidize aromatic hydrocarbons and related compounds to cis-arene diols. These enzymes utilize a mononuclear non-heme iron center to catalyze the addition of dioxygen to their respective substrates. The present study was conducted to provide essential structural information necessary for elucidating the mechanism of action of NDO.nnnRESULTSnThe three-dimensional structure of NDO has been determined at 2.25 A resolution. The molecule is an alpha 3 beta 3 hexamer. The alpha subunit has a beta-sheet domain that contains a Rieske [2Fe-2S] center and a catalytic domain that has a novel fold dominated by an antiparallel nine-stranded beta-pleated sheet against which helices pack. The active site contains a non-heme ferrous ion coordinated by His208, His213, Asp362 (bidentate) and a water molecule. Asn201 is positioned further away, 3.75 A, at the missing axial position of an octahedron. In the Rieske [2Fe-2S] center, one iron is coordinated by Cys81 and Cys101 and the other by His83 and His104.nnnCONCLUSIONSnThe domain structure and iron coordination of the Rieske domain is very similar to that of the cytochrome bc1 domain. The active-site iron center of one of the alpha subunits is directly connected by hydrogen bonds through a single amino acid, Asp205, to the Rieske [2Fe-2S] center in a neighboring alpha subunit. This is likely to be the main route for electron transfer.

Construction and use of a new broad-host-range lacZ transcriptional fusion vector, pHRP309, for Gram − bacteria

A new lacZ transcriptional fusion vector, pHRP309, based on the IncQ plasmid RSF1010, was constructed and shown to be easily mobilized into a variety of Gram- eubacteria. We also developed a two-step cloning procedure to facilitate the cloning of small promoter fragments into the fusion vector. A set of cohort vectors was constructed which allowed directed cloning of fragments downstream from an omega streptomycin/spectinomycin-resistance cassette while maintaining multiple flanking restriction sites. The omega cassette provides a selectable antibiotic-resistance marker for cloning promoters into the fusion vector and makes mapping to determine fragment orientation unnecessary. The presence of the omega cassette also decreases background beta-galactosidase activity by decreasing readthrough transcription from plasmid sequences. The fusion vector carries a gentamicin-resistance-encoding gene as the selectable marker and can therefore be used in Tn5 (kanamycin-resistant) and Tn10 (tetracycline-resistant) mutant strains. Since pHRP309 is a member of the IncQ incompatibility group, it is compatible with IncP cloning vectors and can be used in strains carrying cloned regulatory genes. Using this system, we cloned the positively regulated Pseudomonas putida pcaI promoter and studied its regulation.

Bacterial chemotaxis to pollutants and plant-derived aromatic molecules.

There is accumulating evidence that motile bacteria are chemotactically attracted to environmental pollutants that they can degrade. Chemotaxis, the ability of motile bacteria to detect and respond to specific chemicals in the environment, can increase an organisms chances of locating useful sources of carbon, nitrogen and energy, and could thus play an important role in the biodegradation process. Recent evidence demonstrating that chemotaxis and biodegradation genes are coordinately regulated suggests that these processes are intimately linked in nature.

Cloning and sequencing of the genes encoding 2-nitrotoluene dioxygenase from Pseudomonas sp. JS42

The first step in the metabolism of 2-nitrotoluene (2NT) by Pseudomonas sp. JS42 (JS42) is the addition of dioxygen to the aromatic nucleus of 2NT to form 3-methylcatechol with concomitant release of nitrite. This reaction is catalyzed by the three-component dioxygenase system 2-nitrotoluene 2,3-dioxygenase (2NTDO). We report here the cloning and nucleotide (nt) sequence of a 4912-basepair (bp) SacI DNA fragment from JS42 encoding all of the genes required for 2NTDO activity. Sequence analysis of the 4912-bp SacI DNA fragment revealed five open reading frames (ORFs). The amino acid (aa) sequences of the predicted polypeptides from these ORFs exhibit high homology to the aa sequences of polypeptides from other three-component dioxygenase systems. Based on aa sequence analyses, four of the peptides were designated Reductase2NT, Ferredoxin2NT, ISP alpha 2NT and ISP beta 2NT (ISP for iron-sulfur protein) with gene designations ntdAaAbAcAd. The predicted aa sequence from the remaining ORF (ORF2) had identity to ISP alpha subunits from other three-component dioxygenase systems but had a calculated molecular weight (M(r)) of 21,259, which is uncharacteristically small for ISP alpha subunits.

The role of active-site residues in naphthalene dioxygenase

The three-component naphthalene dioxygenase enzyme system catalyzes the first step in the degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. A member of a large family of bacterial Rieske non-heme iron oxygenases, naphthalene dioxygenase is known to oxidize over 60 different aromatic compounds, and many of the products are enantiomerically pure. The crystal structure of the oxygenase component revealed the enzyme to be an α3β3 hexamer and identified the amino acids located near the active site. Site-directed mutagenesis studies have identified the residues involved in electron transfer and those responsible for controlling the regioselectivity and enantioselectivity of the enzyme. The results of these studies suggest that naphthalene dioxygenase can be engineered to catalyze a new and extended range of useful reactions.

Aromatic Ring Hydroxylating Dioxygenases

Aromatic ring hydroxylating dioxygenases play a key role in the biodegradation of numerous environmental pollutants, both in the natural environment (via natural attenuation) and in the engineered bioremediation systems. Recent structural and mechanistic information, together with enzyme engineering and strain construction strategies should allow the development of engineered microorganisms with new and/or optimized degradation abilities. The continued application of these approaches should also facilitate the development of Rieske non-heme iron dioxygenases with requisite selectivities for specific opportunities in target direct biocatalysis or metabolic engineering.

Multiple mutations at the active site of naphthalene dioxygenase affect regioselectivity and enantioselectivity

The importance of five amino acids at the active site of the multicomponent naphthalene dioxygenase (NDO) system was determined by generating site-directed mutations in various combinations. The substrate specificities of the mutant enzymes were tested with the substrates indole, indoline, 2-nitrotoluene (2NT), naphthalene, biphenyl, and phenanthrene. Transformation of these substrates measured the ability of the mutant enzymes to catalyze dioxygenation, monooxygenation, and desaturation reactions. In addition, the position of oxidation and the enantiomeric composition of products were characterized. All enzymes with up to three amino acid substitutions were able to catalyze dioxygenation reactions. A subset of these enzymes could also catalyze the monooxygenation of 2NT and desaturation of indoline. Single amino acid substitutions at positions 352 and 206 had the most profound effects on product formation. Of the single mutations made, only changes at position 352 affected the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene, but in the presence of the F352I mutation, changes at positions 206 and 295 also affected enantioselectivity. Major shifts in regioselectivity with biphenyl and phenanthrene resulted with several of the singly, doubly, and triply mutated enzymes. A new product not formed by the wild-type enzyme, phenanthrene cis-9,10-dihydrodiol, was formed as a major product from phenanthrene by enzymes with two (A206I/F352I) or three amino acid substitutions (A206I/F352I/H295I). The results indicate that a variety of amino acid substitutions are tolerated at the active site of NDO. Journal of Industrial Microbiology & Biotechnology (2001) 27, 94–103.

Cloning and sequence analysis of a catechol 2,3-dioxygenase gene from the nitrobenzene-degrading strain Comamonas sp JS765

Comamonas sp strain JS765 utilizes nitrobenzene as a carbon and nitrogen source. The initial attack on nitrobenzene is carried out by nitrobenzene 1,2-dioxygenase, which converts nitrobenzene to an unstable nitrohydrodiol that spontaneously decomposes to form catechol and nitrite. Catechol is then degraded via a meta cleavage pathway. We now report the cloning of a DNA fragment carrying a catechol 2,3-dioxygenase gene from JS765. Nucleotide sequence analysis revealed three open reading frames (ORFs) predicted to encode proteins of 33.6, 13.0, and 35.0u2009kDa. Homology searches of the deduced amino acid sequences of three proteins suggested that ORF1 encodes a LysR-type transcriptional regulator, ORF2 encodes a XylT-type ferredoxin, and ORF3 encodes a catechol 2,3-dioxygenase. The putative regulatory gene, designated cdoR, is divergently transcribed from the ferredoxin and catechol dioxygenase genes, cdoT and cdoE, respectively. The catechol 2,3-dioxygenase is most similar in amino acid sequence to the I.2.C subfamily of extradiol dioxygenases which include 3-methylcatechol 2,3-dioxygenase from the aniline- and toluidine-degrading Pseudomonas putida UCC2, TbuE from the toluene monooxygenase pathway of Pseudomonaspickettii PKO1 and catechol 2,3-dioxygenase II from the TOL plasmid pWW15. The substrate range of the catechol 2,3-dioxygenase produced by the recombinant E. coli strains was very similar to that of the enzyme present in nitrobenzene-grown JS765, suggesting that we have cloned the catechol 2,3-dioxygenase gene required for nitrobenzene degradation.

Purification, characterization, and crystallization of the components of a biphenyl dioxygenase system from Sphingobium yanoikuyae B1.

Sphingobium yanoikuyae B1 initiates the catabolism of biphenyl by adding dioxygen to the aromatic nucleus to form (+)-cis-(2R, 3S)-dihydroxy-1-phenylcyclohexa-4,6-diene. The present study focuses on the biphenyl 2,3-dioxygenase system, which catalyzes the dioxygenation reaction. This enzyme has been shown to have a broad substrate range, catalyzing the dioxygenation of not only biphenyl, but also three- and four-ring polycyclic aromatic hydrocarbons. Extracts prepared from biphenyl-grown B1 cells contained three protein components that were required for the oxidation of biphenyl. The genes encoding the three components (bphA4, bphA3 and bphA1f,A2f) were expressed in Escherichia coli. Biotransformations of biphenyl, naphthalene, phenanthrene, and benzo[a]pyrene as substrates using the recombinant E. coli strain resulted in the formation of the expected cis-dihydrodiol products previously shown to be produced by biphenyl-induced strain B1. The three protein components were purified to apparent homogeneity and characterized in detail. The reductase component (bphA4), designated reductaseBPH-B1, was a 43xa0kD monomer containing one mol FAD/mol reductaseBPH-B1. The ferredoxin component (bphA3), designated ferredoxinBPH-B1, was a 12xa0kD monomer containing approximately 2xa0g-atoms each of iron and acid-labile sulfur. The oxygenase component (bphA1f,A2f), designated oxygenaseBPH-B1, was a 217xa0kD heterotrimer consisting of α and β subunits (approximately 51 and 21xa0kD, respectively). The iron and acid-labile sulfur contents of oxygenaseBPH-B1 per αβ were 2.4 and 1.8xa0g-atom per mol, respectively. Reduced ferredoxinBPH-B1 and oxygenaseBPH-B1 each gave EPR signals typical of Rieske [2Fe-2S] proteins. Crystals of reductaseBPH-B1, ferredoxinBPH-B1 and oxygenaseBPH-B1 diffracted to 2.5xa0Å, 2.0xa0Å and 1.75xa0Å, respectively. The structures of the three proteins are currently being determined.