Specificity of Substrate Recognition by Pseudomonas fluorescens N3 Dioxygenase

Abstract

Pseudomonas fluorescens N3 is able to grow on naphthalene as the sole carbon and energy source. The mutant TTC1, blocked at the dihydrodiol dehydrogenase level, which can transform the hydrocarbon into the corresponding dihydrodiol, has been used to produce bioconversion products. To rationalize the different grades of conversion obtained with different substrates, a study was performed using non-naphthalene derivatives, including benzenes, conjugated benzenes, and polycyclic aromatic hydrocarbons. The corresponding diols obtained by bioconversion have been isolated and characterized. A theoretical model that considers both energy and geometry factors has been proposed to rationalize the experimental data. Good agreement has been found between the calculated values and the experimental results.Pseudomonas dioxygenases are a family of closely related enzymes that can add an oxygen molecule to a substrate double bond (1). Their ability to make such a transformation on aromatic compounds, often the first step in the biodegradation of such compounds (2), is particularly interesting. The high stability of aromatic compounds requires an unusually high redox potential that, in most cases, is made available by two or three component enzymes (3). The active site is believed to belong to the class of Rieske-type iron-sulfur proteins, in which the iron, coordinated by two histidine nitrogens and two cysteine sulfurs, is, at the same time, the end point of a long redox chain transporting the necessary electrons from NADH (or NADPH) and the coordination site of molecular oxygen (3). In the general framework of this type of dioxygenase, the specificity of recognition could be related to the geometric and functional characteristics of the active site. As a consequence, there are well-known oxygenases that transform monocyclic compounds (benzene (4) or toluene (5)), naphthalenes (6), or polycyclic aromatic hydrocarbons (7).Since 1992, our research group has been interested in exploiting the power of the dioxygenase of P. fluorescens N3 that we isolated from the activated sludge of a wastewater treatment plant (8). The wild type is able to completely degrade naphthalene and some of its derivatives and to transform many other naphthalenes into the corresponding salicylic acids (8).We later isolated a mutant strain (TTC1) blocked at the dihydrodiol dehydrogenase level (9), and then we cloned the naphthalene dioxygenase gene in Escherichia coli JM109 (10). Thus, we could efficiently produce dihydrodiols from many naphthalenes carrying substituents in both position 1 and position 2. The yield seemed to be correlated to two aspects, the electronic characteristics of the substituents and their position, and was higher for electron releasing groups in position 2. However, the yield trend was not readily understandable, so we decided to extend our investigation to assess the specificity of N3 dioxygenase.Two strongly correlated actions were considered: the experimental verification of substrate specificity (by testing diverse aromatic s...

Efficient polycyclic aromatic hydrocarbons dihydroxylation in direct micellar systems

“…The enantioselective oxidation of naphthalene and anthracene catalyzed by the E. coli JM109 (pPS1778) recombinant strain carrying naphthalene dioxygenase and regulatory genes cloned from P. fluorescens N3 generates cis-(1R,2S)-1,2-dihydro-1,2-dihydroxynaphthalene and cis-(1R,2S)-1,2dihydro-1,2-dihydroxyanthracene, respectively, whereas phenanthrene conversion yields two products: cis-(3S,4R)-3,4-dihydro-3,4-dihydroxyphenanthrene and cis-(1R,2S)-1,2-dihydro-1,2-dihydroxyphenanthrene at a ratio of 5.2:1, as previously reported (Di Gennaro et al, 1997c).…”

“…The purpose of the present study is to optimizate the production of cis-dihydroxydihydro compounds from other, less water-soluble polycyclic aromatic hydrocarbons, such as anthracene ("A" in Scheme 1) and phenanthrene ("B" in Scheme 1). Such substances have been reported to be converted by the naphthalene dioxygenase employed in the present study, but the biotransformation resulted in very low final yields (Di Gennaro et al, 1997c). The production of large quantities of such derivatives will allow for the design and synthesis of optically pure new compounds of potential industrial and pharmaceutical interest.…”

“…The E. coli JM109 (pPS1778) recombinant strain carrying naphthalene dioxygenase and regulatory genes cloned from P. fluorescens N3 was used for bioconversion studies Di Gennaro et al, 1997a, 1997b. This biological system is able to catalyze the conversion of naphthalene and other polycyclic aromatic compounds to the corresponding cis,cis-dihydrodihydroxylated derivatives as previously reported (Bestetti et al, 1994(Bestetti et al, , 1995Di Gennaro et al, 1997a, 1997b, 1997c.…”

Products from the Incomplete Metabolism of Pyrene by Polycyclic Aromatic Hydrocarbon-Degrading Bacteria

“…It also has been found in pyrene-contaminated marine sediments (30) and has accumulated when pyrene was added to enrichment cultures derived from the sediment (29). The formation of a cis-dihydrodiol from pyrene is consistent with its oxidation via a PAH dioxygenase, an enzyme class for which examples from several bacteria are known to have broad substrate ranges (14,15,18,27,28,34,37,46).…”

Substrate Specificity of Naphthalene Dioxygenase: Effect of Specific Amino Acids at the Active Site of the Enzyme

“…Recently, NDO from Pseudomonas putida G7 was shown to produce a minor product (3%) from biphenyl that was identified by GC-MS and indirect methods as biphenyl 3,4-dihydrodiol (3). NDO from Pseudomonas fluorescens N3 was also reported to form a small amount (15%) of cis-biphenyl 3,4-dihydrodiol (13). However, the low yield of product and reported instability of the compound precluded extensive characterization.…”