Metabolism of naphthalene and methylnaphthalenes in Pseudomonas putida : Elucidation of catabolic pathways and purification and properties of 1,2-Dihydroxynaphthalene dioxygenase

Abstract

I. Polycyclic aromatic hydrocarbons (PAHs), which include naphthalene and alkyl-substituted naphthalenes, are universal products of combustion of organic matter. The mutagenic and carcinogenic nature of several PAHs makes studies on various aspects of these compounds extremely important. The genetics and metabolism of naphthalene degradation in pseudomonads have been studied in fine detail. The first two enzymes of the naphthalene catabolic pathway, namely naphthalene dioxygenase and cis-naphthalene dihydrodiol dehydrogenase, have been purified and characterized. However, little attention has been paid to the comparative study of the metabolism of naphthalene and alkyl-substituted naphthalenes in a single bacterium. Also, much more remains to be known about 1,2-dihydroxynaphthalene dioxygenase—a first ring-opening dioxygenase in the naphthalene catabolic pathway. II. Keeping these aspects in mind, a bacterium has been isolated from the soil samples near a petrol station by enrichment culture technique. It grows on naphthalene and 2-methylnaphthalene as sole sources of carbon and energy. When adapted, it also grows on 1-methylnaphthalene as sole source of carbon and energy. On the basis of various morphological, physiological and biochemical tests, the organism has been identified as Pseudomonas putida. It attains full growth at 17 h, 28 h and 24 h in the presence of naphthalene, 1-methylnaphthalene and 2-methylnaphthalene, respectively. III. In order to deduce the pathways for the catabolism of naphthalene, 1- and 2-methylnaphthalene, the metabolites have been isolated from the spent medium and purified by thin-layer chromatography. Emphasis has been placed on the structural characterization of isolated metabolites by GC-MS, NMR and IR spectroscopy, demonstration of enzyme activities in cell-free extracts and measurement of oxygen uptake by whole cells in the presence of various probable metabolic intermediates. The results obtained from such a study are described below: (1) On the basis of isolation of catechol from the spent medium and its structure determination by mass spectrometry and demonstration of the activities of most of the enzymes of the naphthalene catabolic pathway, the degradation of naphthalene in this organism has been shown to follow the same path reported earlier by several workers. The catabolism of naphthalene is initiated by double hydroxylation of one of the aromatic rings to form cis-dihydrodiol naphthalene, which is converted to 1,2-dihydroxynaphthalene by a dehydrogenase. The third step in the degradation of naphthalene is the cleavage of the hydroxylated ring of 1,2-dihydroxynaphthalene to 2-hydroxychromene-2-carboxylate, which is further metabolized by a series of enzyme-catalyzed reactions to form salicylate and catechol as end products. Detection of catechol 2,3-dioxygenase activity in the cell-free extract shows that catechol is further metabolized by the meta pathway. (2) On the basis of the isolation of metabolites and their structural characterization, occurrence of two pathways for the catabolism of 1-methylnaphthalene has been shown. In one of the pathways, the methyl group of 1-methylnaphthalene remains intact while it is being metabolized by oxidation of the aromatic ring. The oxidation of 1-methylnaphthalene is initiated by double hydroxylation of the aromatic ring adjacent to the one bearing the methyl moiety, giving rise to cis-1,2-dihydroxy-1,2-dihydro-8-methylnaphthalene. This compound is converted to 3-methyl salicylate and 3-methylcatechol by a route analogous to that for the oxidation of naphthalene. The 3-methylcatechol is further metabolized by the meta pathway. (3) Detection of 1-hydroxymethylnaphthalene in the spent medium indicates the alternate mode of oxidation of 1-methylnaphthalene by the oxidation of the methyl substituent. The 1-hydroxymethylnaphthalene is further converted to 1-naphthoic acid via 1-naphthaldehyde. The failure of the bacterium to grow on either 1- or 2-naphthoic acid or to respire in their presence suggests that 1-naphthoic acid is the end product of this pathway. (4) The occurrence of two modes of oxidation of 1-methylnaphthalene in this organism can be explained as follows: The organism grows on 1-methylnaphthalene as sole source of carbon and energy by the oxidation of aromatic rings in one pathway, while the excess of 1-methylnaphthalene accumulated in the organism might be removed out of the system by making it water-soluble by side-chain oxidation to form 1-naphthoic acid. (5) Detection of 4-methyl salicylate in the spent medium indicates that the initial step in the oxidation of 2-methylnaphthalene could be the double hydroxylation of the aromatic ring to give rise to cis-1,2-dihydroxy-1,2-dihydro-7-methylnaphthalene, which after a series of enzyme-catalyzed reactions is converted to 4-methyl salicylate and 4-methylcatechol as end products. (6) Alternatively, 2-methylnaphthalene may undergo side-chain hydroxylation to form 2-hydroxymethylnaphthalene, which in turn could be oxidized in two ways: (i) The hydroxymethyl group could be further oxidized to aldehyde and acid to form 2-naphthoic acid as the end product. Evidence for this comes from the isolation of 2-naphthoic acid from the spent medium. (ii) Detection of 7-hydroxymethyl-1,2-naphthoquinone and 4-hydroxymethyl salicylaldehyde in the spent medium suggests that the 2-hydroxymethylnaphthalene may also be oxidized by double hydroxylation of the aromatic ring to form cis-1,2-dihydroxy-1,2-dihydro-7-hydroxymethylnaphthalene, which is further oxidized by a similar pathway as seen in the case of naphthalene. The resultant products of this pathway could be 4-hydroxymethyl salicylate and 4-hydroxymethylcatechol. However, these compounds did not accumulate in the spent medium. Detection of catechol 2,3 a dioxygenase activity in the cell-free extract of 2-methylnaphthalene-grown cells suggests that 4-hydroxy-2-methylcatechol could be further metabolized by the meta pathway. (7) The activities of catechol 2,3-dioxygenase, salicylate hydroxylase, cis-naphthalene dihydrodiol dehydrogenase remain the same in the cell-free extracts of naphthalene- and 2-methylnaphthalene-grown cells. Likewise, the rate of oxygen uptake by whole cells in the presence of salicylate, catechol and methylcatechols remains unaltered. Thus, it appears that a non-specific set of enzymes is involved in the degradation of naphthalene, 2-methylnaphthalene and 2-hydroxymethylnaphthalene. IV. A successful attempt has been made to purify 1,2-dihydroxynaphthalene dioxygenase by affinity chromatography. It was expected that ?-naphthol, an analog of 1,2-dihydroxynaphthalene, could be used to prepare the affinity matrix for the purification of the enzyme. 3-naphthol was coupled to benzidyl Sepharose 4B-CL by diazotization reaction. The matrix thus obtained was blood red in color. The enzyme bound on this column could be eluted with 15 mM 3-naphthol in 50 mM phosphate buffer pH 7.5, containing 10% glycerol (v/v) and 10% ethanol (v/v). This buffer is referred to as PGE. Before loading on the affinity column, the crude extract was passed through a DEAE-Sephacel column. The affinity-purified enzyme was highly pure. It moved on 7.5% PAGE and 10% SDS-PAGE as a single homogeneous band. Some of the properties of the enzyme are described as follows: (1) The purified 1,2-dihydroxynaphthalene dioxygenase is stable at 4°C for 8 days in the presence of 10% glycerol (v/v), 10% ethanol (v/v), in 50 mM sodium phosphate buffer pH 7.5 (PGE). In the absence of organic solvents, the enzyme loses 60% of its activity in 24 h. (2) It has been observed that the enzyme at different purification steps is active only if external iron is added to it under anaerobic conditions. The enzyme also loses its activity upon dialysis. The inactivated enzyme could be fully reactivated by incubating it in the presence of 0.05 mM FeSO? under anaerobic conditions for 1 h. (3) Apart from the requirement for added iron, the enzyme also contains bound iron. It is estimated to be one per subunit. (4) The native molecular weight (as determined by gel filtration chromatography on a Sephacryl S-300 column) and subunit molecular weight of the enzyme (as determined by SDS-PAGE) are estimated to be approximately 316 kDa and 32 kDa respectively. Thus, it looks like the enzyme is a decamer of 10 identical subunits. (5) The specific activity of the enzyme at pH 5.5 with 1,2-dihydroxynaphthalene as a substrate is calculated to be 10,200. It also acts on catechol and 4-methylcatechol. At pH 7.5, the enzyme is equally active with catechol and 4-methylcatechol, their specific activities being 260 and 225, respectively. The enzyme does not act on 2,3- and 3,4-dihydroxybenzoic acid and 3-methylcatechol is a poor substrate. The Km values of the enzyme for catechol and 4-methylcatechol are 2 ?M and 1.72 mM, respectively. (6) When catechol, 4-methylcatechol and 3-methylcatechol are used as substrates, the reaction products formed are yellow in color with absorption maxima at 375 nm, 382 nm and 302 and 390 nm, respectively, indicating these to be semialdehydes. The nature of the reaction products formed suggests that 1,2-dihydroxynaphthalene dioxygenase opens the aromatic ring adjacent to the carbon atoms bearing hydroxyl groups. (7) The enzyme is maximally active at 30°C and it is optimally active in the broad pH range of 7.5–8.5. (8) The enzyme is strongly inhibited by heavy metal ions, metal chelators, sulfhydryl compounds, sulfhydryl reagents and denaturing reagents. It is particularly noteworthy that the sulfhydryl compounds such as 2-ME, DTT, reduced glutathione and cysteine, which are used to stabilize many enzymes, strongly inhibit this enzyme. (9) ?-naphthol, 3-naphthol and 3-methylcatechol (a poor substrate) are competitive inhibitors of the enzyme, and their Ki values are calculated to be 23 ?M, 38 ?M and 5 ?M, respectively. (10) The purified enzyme is colorless and does not absorb in the visible range. However, it absorbs in the UV region with absorption maximum at 280 nm. The fluorescence spectrum of the enzyme has been recorded by fixing the excitation maximum at 280 nm. The emission maximum of the enzyme is 336 nm. This fluorescence does not change appreciably in a pH range of 7.0–9.5. It indicates that most probably, the fluorescence of the enzyme is due to tryptophan residues. The far UV-CD spectrum of the enzyme shows two troughs at 210 nm and 205 nm, characteristic of an ?-helical structure. The N-terminal 21 amino acids of the enzyme have been sequenced. This sequence shows a near 100% homology with the N-terminal sequence deduced from the nucleotide sequence of 1,2-dihydroxynaphthalene dioxygenase gene nahC. Amino acid analysis of the enzyme shows that it is rich in glycine and acidic amino acids and low in cysteine and methionine. (12) At pH 8.5 and above, the enzyme has been shown to cyclize the reaction products of 4-methylcatechol and 3-methylcatechol to 4-methyl-1,2-benzoquinone and 2-hydroxy-6-methylpyran-2-carboxylate, respectively. The enzyme does not cyclize the reaction product of catechol. Using various enzyme inhibitors like 2-ME, HgCl?, ?-naphthol etc. and by heat inactivation of the enzyme, the cyclization of the ring-opened products of methylcatechols has been shown to be mediated by the enzyme. (13) It has been postulated by several investigators that 2-hydroxychromene-2-carboxylate (the product of the ring cleavage of 1,2-dihydroxynaphthalene) is formed as a consequence of ring fission of 1,2-dihydroxynaphthalene followed by cyclization before the release of the product from the enzyme. (14) Experimental proof is given for the existence of a cyclization mechanism on this enzyme. This cyclization occurs when the dioxygenase reaction is complete. Thus, it appears that ring cleavage and cyclization do not occur sequentially on the enzyme. Instead, after dioxygenase reaction, the ring-opened product is released from the enzyme. When the dioxygenase reaction is complete, the ring-opened product is cyclized. Thus, the ring opening and cyclization occur independently and are catalyzed by the same enzyme. (15) Amino acids at the active site of the enzyme have been identified using specific modification reagents. DEPC, phenylglyoxal and pHMB have been used to specifically modify histidine, arginine and cysteine, respectively. The modification of the enzyme by these reagents is time and concentration dependent. The loss of enzyme activity upon modification follows pseudo-first-order kinetics. ?-naphthol and 3-naphthol protect the enzyme against modification. (16) Modification kinetics with all the three modification reagents and protection offered by ?- and 3-naphthol against the modification suggest the presence of one histidine, one arginine and one cysteine at or near the active site of the enzyme. The possible role of these amino acids in the enzyme catalysis is discussed. In conclusion, this thesis describes the isolation and identification of a bacterium (Pseudomonas putida) which could grow on naphthalene and methylnaphthalenes as sole sources of carbon and energy, elucidation of catabolic pathways for naphthalene, 1- and 2-methylnaphthalene, affinity purification of 1,2-dihydroxynaphthalene dioxygenase and some of its structural, physicochemical, kinetic and mechanistic properties, involvement of one each of histidine, arginine and cysteine at or near the active site of the enzyme. The identity of the enzyme as 1,2-dihydroxynaphthalene dioxygenase has been established by N-terminal sequence homology with the same enzyme from another source.