Regiospecific O-methylation of naphthoic acids catalyzed by NcsB1, an O-methyltransferase involved in the biosynthesis of the enediyne antitumor antibiotic neocarzinostatin

Neocarzinostatin, a clinical anticancer drug, is the archetypal member of the chromoprotein family of enediyne antitumor antibiotics that are composed of a nonprotein chromophore and an apoprotein. The neocarzinostatin chromophore consists of a nine-membered enediyne core, a deoxyaminosugar, and a naphthoic acid moiety. We have previously cloned and sequenced the neocarzinostatin biosynthetic gene cluster and proposed that the biosynthesis of the naphthoic acid moiety and its incorporation into the neocarzinostatin chromophore are catalyzed by five enzymes NcsB, NcsB1, NcsB2, NcsB3, and NcsB4. Here we report the biochemical characterization of NcsB1, unveiling that: (i) NcsB1 is an S-adenosyl-L-methionine-dependent O-methyltransferase; (ii) NcsB1 catalyzes regiospecific methylation at the 7-hydroxy group of its native substrate, 2,7-dihydroxy-5-methyl-1-naphthoic acid; (iii) NcsB1 also recognizes other dihydroxynaphthoic acids as substrates and catalyzes regiospecific O-methylation; and (iv) the carboxylate and its ortho-hydroxy groups of the substrate appear to be crucial for NcsB1 substrate recognition and binding, and O-methylation takes place only at the free hydroxy group of these dihydroxynaphthoic acids. These findings establish that NcsB1 catalyzes the third step in the biosynthesis of the naphthoic acid moiety of the neocarzinostatin chromophore and further support the early proposal for the biosynthesis of the naphthoic acid and its incorporation into the neocarzinostatin chromophore with free naphthoic acids serving as intermediates. NcsB1 represents another opportunity that can now be exploited to produce novel neocarzinostatin analogs by engineering neocarzinostatin biosynthesis or applying directed biosynthesis strategies.     

Molecular models of neocarzinostatin damage of DNA: analysis of sequence dependence in 5''GAGCG:5''CGCTC.

Model building and molecular mechanics and dynamics calculations have been performed on a number of complexes of the post-activated form of the neocarzinostatin chromophore (NCS) with the B-DNA oligomer 5'GAGCG:5'CGCTC. Stable structures with the naphthoic acid moiety intercalated at all base pairs can be constructed. The observed bistranded lesions consisting of an abasic site at the Cyt residue in AGC and a direct break at the Thy residue on the complementary strand can be explained by assuming that NCS in the (R,R) form intercalates between the Ade2-Thy9/Gua3-Cyt8 base step with its 'diradical' core oriented towards the 3'-end of the (+) strand. Sites at C5', C4' and C1' in the minor groove are within a short enough distance from the two radical centers on NCS to permit hydrogen atom abstraction and the formation of the bistranded lesions. Strand cleavage at Thy9 may occur as a single lesion if NCS is intercalated into the Gua3-Cyt8/Cyt4-Gua7 base step with its active core towards the 3'-end of the (-) strand. The results are analyzed, and the utility and limitations of this type of model building are discussed.     

A genomics-guided approach for discovering and expressing cryptic metabolic pathways

Genome analysis of actinomycetes has revealed the presence of numerous cryptic gene clusters encoding putative natural products. These loci remain dormant until appropriate chemical or physical signals induce their expression. Here we demonstrate the use of a high-throughput genome scanning method to detect and analyze gene clusters involved in natural-product biosynthesis. This method was applied to uncover biosynthetic pathways encoding enediyne antitumor antibiotics in a variety of actinomycetes. Comparative analysis of five biosynthetic loci representative of the major structural classes of enediynes reveals the presence of a conserved cassette of five genes that includes a novel family of polyketide synthase (PKS). The enediyne PKS (PKSE) is proposed to be involved in the formation of the highly reactive chromophore ring structure (or "warhead") found in all enediynes. Genome scanning analysis indicates that the enediyne warhead cassette is widely dispersed among actinomycetes. We show that selective growth conditions can induce the expression of these loci, suggesting that the range of enediyne natural products may be much greater than previously thought. This technology can be used to increase the scope and diversity of natural-product discovery.     

Molecular dynamics simulations investigation of neocarzinostatin chromophore-releasing pathways from the holo-NCS protein

The enediyne ring chromophore with strong DNA cleavage activity of neocarzinostatin is labile and therefore stabilization by forming the complex (carrying protein + chromophore: holo-NCS). Holo-NCS has gained much attention in clinical use as well as for drug delivery systems, but the chromophore-releasing mechanism to trigger binding to the target DNA with high affinity and producing DNA damage remain unclear. Three possible pathways were initially determined by conventional MD, essential dynamics and essential dynamics sampling. One of the paths runs along the naphthoate moiety; another runs along the amino sugar moiety; the third along the enediyne ring. Further, calculated forces and time by FPMD (force-probe molecular dynamics) suggest that the opening of the naphthoate moiety is most favorable pathway and Leu45, Phe76 and Phe78 all are key residues for chromophore release. In addition, conformational analyses indicate that the chromophore release is only local motions for the protein.     

Neocarzinostatin: Chemical characterization and partial structure of the non-protein chromophore

The molecular formula C₃₅H₃₅NO₁₂ (mol.wt. 661) is proposed for the biologically active chromophoric component of neocarzinostatin. The partial structure $\text{2}$ is proposed based on ${}^1\text{H}$ NMR and mass spectral data and consists, in part, of a 2,6-dideoxy-2-methylamino-galactose moiety and a naphthoic acid derivative. Special treatments required to obtain spectral data of the labile chromophore are described.     

The biosynthetic gene cluster for the 26-membered ring polyene macrolide pimaricin. A new polyketide synthase organization encoded by two subclusters separated by functionalization genes

The biosynthetic gene cluster for the 26-membered ring of the polyene macrolide pimaricin extends for about 110 kilobase pairs of contiguous DNA in the genome of Streptomyces natalensis. Two sets of polyketide synthase (PKS) genes are separated by a group of small polyketide-functionalizing genes. Two of the polyketide synthase genes, pimS0 and pimS1, have been fully sequenced and disrupted proving the involvement of each of these genes in pimaricin biosynthesis. The pimS0 gene encodes a relatively small acetate-activating PKS (approximately 193 kDa) that appears to work as a loading protein which "presents" the starter unit to the second PKS subunit. The pimS1 gene encodes a giant multienzyme (approximately 710 kDa) harboring 15 activities responsible for the first four cycles of chain elongation in pimaricin biosynthesis, resulting in formation of the polyene chromophore.     

The product of dpsC confers starter unit fidelity upon the daunorubicin polyketide synthase of Streptomyces sp. strain C5

The biosynthesis of daunorubicin and its precursors proceeds via the condensation of nine C-2 units derived from malonyl-CoA onto a propionyl starter moiety. The daunorubicin polyketide biosynthesis gene cluster of Streptomyces sp. strain C5 has two unique open reading frames, dpsC and dpsD, encoding, respectively, a fatty acid ketoacyl synthase (KAS) III homologue that is lacking an active-site cysteine and a proposed acyl-CoA:acyl carrier protein acyltransferase. The two genes are positioned directly downstream of dpsA and dpsB which encode the alpha and beta components of the type II KAS, respectively. Expression of the dpsABCDEFGdauGI genes in Streptomyces lividans resulted in the formation of aklanonic acid, the first stable chromophore of the daunorubicin biosynthesis pathway. Deletion of dpsC, but not dpsD, from this gene set resulted in the formation of desmethylaklanonic acid, derived from an acetyl-CoA starter unit, and aklanonic acid, derived from propionyl-CoA, in a 60:40 ratio. Thus, DpsC contributes to the selection of propionyl-CoA as the starter unit but does not alone dictate it. A dpsCD deletion mutant of Streptomyces sp. strain C5 (C5VR5) still produced daunorubicin but, more significantly, anthracycline and anthracyclinone derivatives resulting from the use of acetyl-CoA as an alternative starter moiety. Expression of dpsC, but not dpsD, in mutant C5VR5 restored the wild-type phenotype. Among the new compounds was the new biosynthesis product feudomycin D. These results suggest that in the absence of DpsC, the daunorubicin PKS complex behaves promiscuously, utilizing both acetyl-CoA (ca. 60% of the time) and propionyl-CoA (ca. 40%) as starter units. The fact that DpsC is not required for initiation with propionyl-CoA is significant, as the information must then lie in other components of the PKS complex. We propose to call DpsC the propionyl starter unit "fidelity factor." Copyright 2001 Academic Press.     

Binding of the nonprotein chromophore of neocarzinostatin to deoxyribonucleic acid

The methanol-extracted, nonprotein chromophore of neocarzinostatin (NCS), which has DNA-degrading activity comparable to that of the native antibiotic, was found to have a strong affinity for DNA. Binding of chromophore was shown by (1) quenching by DNA of the 440-nm fluorescence and shifting of the emission peak to 420 nm, (2) protection by DNA against spontaneous loss of activity in aqueous solution, and (3) inhibition by DNA of the spontaneous generation of 490-nm fluorescence. Good quantitative correlation was found between these three methods in measuring chromophore binding. There was nearly a 1:1 correspondence between loss of chromophore activity and generation of 490-nm fluorescence, suggesting spontaneous degradation of active chromophore to a highly fluorescent product. Chromophore showed a preference for DNA high in adenine + thymine content in both fluorescence quenching and protection studies. NCS apoprotein, which is known to bind and protect active chromophore, quenched the 440-nm fluorescence, shifted the emission peak to 420 nm, and inhibited the generation of 490-nm fluorescence. Chromophore had a higher affinity for apoprotein than for DNA. Pretreatment of chromophore with 2-mercaptoethanol increased the 440-nm fluorescence seven-fold and eliminated the tendency to generate 490-nm fluorescence. The 440-nm fluorescence of this inactive material was also quenched by DNA and shifted to 420 nm, indicating an affinity for DNA comparable to that of untreated chromophore. However, its affinity for apoprotein was much lower than that of untreated chromophore. Both 2-mercapto-ethanol-treated and untreated chromophore unwound supercoiled pMB9 DNA, suggesting intercalation by both molecules. Since no physical evidence for interaction of native neocarzinostatin with DNA has been found, it is likely that dissociation of the chromophore from the protein and association with DNA are important steps in degradation of DNA by neocarzinostatin.     

Chimeric 6-methylsalicylic acid synthase with domains of acyl carrier protein and methyltransferase from Pseudallescheria boydii shows novel biosynthetic activity

Polyketides are important secondary metabolites, many of which exhibit potent pharmacological applications. Biosynthesis of polyketides is carried out by a single polyketide synthase (PKS) or multiple PKSs in successive elongations of enzyme-bound intermediates related to fatty acid biosynthesis. The polyketide gene PKS306 from Pseudallescheria boydii NTOU2362 containing domains of ketosynthase (KS), acyltransferase (AT), dehydratase (DH), acyl carrier protein (ACP) and methyltransferase (MT) was cloned in an attempt to produce novel chemical compounds, and this PKS harbouring green fluorescent protein (GFP) was expressed in Saccharomyces cerevisiae. Although fluorescence of GFP and fusion protein analysed by anti-GFP antibody were observed, no novel compound was detected. 6-methylsalicylic acid synthase (6MSAS) was then used as a template and engineered with PKS306 by combinatorial fusion. The chimeric PKS containing domains of KS, AT, DH and ketoreductase (KR) from 6MSAS with ACP and MT from PKS306 demonstrated biosynthesis of a novel compound. The compound was identified with a deduced chemical formula of C₇H₁₀O₃, and the chemical structure was named as 2-hydroxy-2-(propan-2-yl) cyclobutane-1,3-dione. The novel compound synthesized by the chimeric PKS in this study demonstrates the feasibility of combinatorial fusion of PKS genes to produce novel polyketides.     

Affinity transfer by CDR grafting on a nonimmunoglobulin scaffold

Neocarzinostatin (NCS) is a small "all beta" protein displaying the same overall fold as immunoglobulins. This protein possesses a well-defined hydrophobic core and two loops structurally equivalent to the CDR1 and CDR3 of immunoglobulins. NCS is the most studied member of the enediynechromoprotein family, and is clinically used as an antitumoral agent. NCS has promise as a drug delivery vehicle if new binding specificities could be conferred on its protein scaffold. Previous studies have shown that the binding specificity of the crevasse can be extended to compounds completely unrelated to the natural enediyne chromophore family. We show here that it is possible to introduce new interaction capacities to obtain a protein useful for drug targeting by modifying the immunoglobulin CDR-like loops. We transferred the CDR3 of the VHH chain of camel antilysozyme immunoglobulin to the equivalent site in the corresponding loop of neocarzinostatin. We then evaluated the stability of the resulting structure and its affinity for lysozyme. The engineered NCS-CDR3 presents a structure similar to that of the wild-type NCS, and is stable and efficiently produced. ELISA, ITC, and SPR measurements demonstrated that the new NCS-CDR3 specifically bound lysozyme.     

Biodegradation Kinetics of Phenanthrene by a Fusant Strain

The fusant strain (F14), which produced by protoplast fusion between Sphingomonas sp. GY2B (GenBank DQ139343) and Pseudomonas sp. GP3A (GenBank EU233280), was tested for phenanthrene biodegradation at 30 °C and pH of 7.0. The kinetics of phenanthrene biodegradation by F14 was investigated over a wide range of initial concentration (15-1,000 mg l(-1)). The rate and the extent of phenanthrene degradation increased with the increase of concentration up to 230 mg l(-1), which indicated negligible inhibition effect at low concentrations. The non-competitive inhibition model was found to be fit for the process. GC-MS analysis showed that biodegradation of phenanthrene by F14 was via dioxygenation at both 1,2- and 3,4-positions and followed by 2-hydroxy-1-naphthoic acid and 1-hydroxy-2-naphthoic acid. The relative intensity of 2-hydroxy-1-naphthoic acid was approximately 3-4 times higher than that of 1-hydroxy-2-naphthoic acid, indicating the 2-hydroxy-1-naphthoic acid was the predominant product in the phenanthrene degradation by fusant strain F14.     

Transformation of 1- and 2-methylnaphthalene by Cunninghamella elegans.

Cunninghamella elegans metabolized 1- and 2-methylnaphthalene primarily at the methyl group to form 1- and 2-hydroxymethylnaphthalene, respectively. Other compounds isolated and identified were 1- and 2-naphthoic acids, 5-hydroxy-1-naphthoic acid, 5-hydroxy-2-naphthoic acid, 6-hydroxy-2-naphthoic acid, and phenolic derivatives of 1- and 2-methylnaphthalene. The metabolites were isolated by thin-layer and reverse-phase high-pressure liquid chromatography and characterized by the application of UV-visible absorption, 1H nuclear magnetic resonance, and mass spectral techniques. Experiments with [8-14C]2-methylnaphthalene indicated that over a 72-h period, 9.8% of 2-methylnaphthalene was oxidized to metabolic products. The ratio of organic-soluble in water-soluble metabolites at 2 h was 92:8, and at 72 h it was 41:59. Enzymatic treatment of the 48-h aqueous phase with either beta-glucuronidase or arylsulfatase released 60% of the metabolites of 2-methylnaphthalene that were extractable with ethyl acetate. In both cases, the major conjugates released were 5-hydroxy-2-naphthoic acid and 6-hydroxy-2-naphthoic acid. The ratio of the water-soluble glucuronide conjugates to sulfate conjugates was 1:1. Incubation of C. elegans with 2-methylnaphthalene under an 18O2 atmosphere and subsequent mass spectral analysis of 2-hydroxymethylnaphthalene indicated that hydroxylation of the methyl group is catalyzed by a monooxygenase.     

Cloning, sequencing and characterization of the biosynthetic gene cluster of sanglifehrin A, a potent cyclophilin inhibitor

Sanglifehrin A (SFA), a potent cyclophilin inhibitor produced by Streptomyces flaveolus DSM 9954, bears a unique [5.5] spirolactam moiety conjugated with a 22-membered, highly functionalized macrolide through a linear carbon chain. SFA displays a diverse range of biological activities and offers significant therapeutic potential. However, the structural complexity of SFA poses a tremendous challenge for new analogue development via chemical synthesis. Based on a rational prediction of its biosynthetic origin, herein we report the cloning, sequencing and characterization of the gene cluster responsible for SFA biosynthesis. Analysis of the 92 776 bp contiguous DNA region reveals a mixed polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) pathway which includes a variety of unique features for unusual PKS and NRPS building block formation. Our findings suggest that SFA biosynthesis requires a crotonyl-CoA reductase/carboxylase (CCR) for generation of the putative unusual PKS starter unit (2R)-2-ethylmalonamyl-CoA, an iterative type I PKS for the putative atypical extender unit (2S)-2-(2-oxo-butyl)malonyl-CoA and a phenylalanine hydroxylase for the NRPS extender unit (2S)-m-tyrosine. A spontaneous ketalization of significant note, may trigger spirolactam formation in a stereo-selective manner. This study provides a framework for the application of combinatorial biosynthesis methods in order to expand the structural diversity of SFA.     

Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid-type III polyketide synthase

Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two approximately 3,000-residue D. discoideum proteins, termed 'steely', as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis.     

Nitroxide-mediated protection against X-ray- and neocarzinostatin-induced DNA damage.

The stable free radical Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidinyloxy) has been shown to protect against X-ray-induced cytotoxicity and hydrogen peroxide- or xanthine oxidase-induced cytotoxicity and mutagenicity. The ability of Tempol to protect against X-ray- or neocarzinostatin (NCS)-induced mutagenicity or DNA double-strand breaks (dsb) was studied in Chinese hamster cells. Tempol (50 mM) provided a protection factor of 2.7 against X-ray-induced mutagenicity in Chinese hamster ovary (CHO) AS52 cells, with a protection factor against cytotoxicity of 3.5. Using the field inversion gel electrophoresis technique of measuring DNA dsb, 50 mM Tempol provides a threefold reduction in DNA damage at an X-ray dose of 40 Gy. For NCS-induced damage, Tempol increased survival from 9% to 80% at 60 ng/mL NCS and reduced mutation induction by a factor of approximately 3. DNA dsb were reduced by a factor of approximately 7 at 500 ng/mL NCS. Tempol is representative of a class of stable nitroxide free radical compounds that have superoxide dismutase-mimetic activity, can oxidize metal ions such as ferrous iron that are complexed to DNA, and may also detoxify radiation-induced organoperoxide radicals by competitive scvenging. The NCS chromophore is reduced by sulfhydryls to an active form. Electron spin resonance (ESR) spectroscopy shows that 2-mercaptoethanol-activated NCS reacts with Tempol 3.5 times faster than does unactivated NCS. Thus, Tempol appears to inactivate the NCS chromophore before a substantial amount of DNA damage occurs.     

Strand scission of deoxyribonucleic acid by neocarzinostatin, auromomycin, and bleomycin: studies on base release and nucleotide sequence specificity

The nucleotide sequence specificity of neocarzinostatin (NCS), auromomycin (AUR), bleomycin (Blm), phleomycin (Phlm), and tallysomycin (Tlm) has been determined by using these antibiotics and their associated chromophores to create strand scissions in end-labeled restriction fragments of DNA and then determining the base sequence of the oligonucleotides formed. NCS and the NCS chromophore induce similar patterns of cleavage in DNA fragments labeled at the 5' terminus. The pattern produced by the AUR chromophore also resembles that of its holoantibiotic. Dithiothreitol enhances the rate of cleavage of DNA by the AUR chromophore but does not alter the sequence specificity. The results suggest that the polypeptide component of AUR and NCS serves primarily as a carrier for the chromophore. When tested with a fragment labeled at the 3' terminus, the products of NCS and AUR cleavage do not display the patterns of chemically produced oligonucleotides cleaved at phosphodiester bonds, suggesting that the 5' terminus is modified by a sugar fragment. NCS primarily attacks thymine (75% of the total bases attacked) and, to a lesser extent, adenine (19%) and cytosine (6%). AUR preferentially attacks guanine (67% of total bases), while attacking less often thymine (24%) and adenine (9%). Bleomycin and its analogues preferentially cleave purine--pyrimidine (5' leads to 3') and pyrimidine--pyrimidine (3' leads to 5') sequences. All (5' leads to 3') GT and GC sequences were cleaved. Phlm G and Phlm-Pep are less active than bleomycin toward purines while Tlm was more active. The patterns of cleavage produced by Blm A2 and Blm B6 are similar, while those produced by Phlm-Pep, Phlm G, Blm-B1', and Blm-Pep resemble one another. The cleavage pattern of Tlm shows quantitative differences from the other analogues tested. Differences between bleomycin and its analogues may be related to structural differences in these molecules.     

Stabilization of neocarzinostatin nonprotein chromophore activity by interaction with apoprotein and with HeLa cells

The methanol-extracted, nonprotein chromophore of the protein antibiotic neocarzinostatin (NCS), which possesses the full in vitro and in vivo deoxyribonucleic acid (DNA) strand-breaking activities and the ability to inhibit DNA synthesis and growth in HeLa cells of the holoantibiotic, is much more labile to inactivation by heat, 2-mercaptoethanol, long-wavelength UV light, and pH values above 4.8. Inactivation is inversely related to the methanol concentration. The pH activity profile of the isolated chromophore extends to pH values below 7.0. Chromophore inactivation is specifically blocked by the apoprotein of NCS; 100-fold higher concentrations of the apoprotein of another protein antibiotic, auromomycin, gave similar protection, whereas bovine serum albumin is even less effective. The chromophore, and not the apoprotein, is inactivated by heat or light (360 nm) as determined by both activity and isoelectric focusing experiments. In contrast to other chromophoric antibiotic substances (daunorubicin and the extracted chromophore of aurodomomycin), the NCS chromophore interacts irreversibly with HeLa cells at 0 degrees C in serum-free medium so as to inhibit subsequent DNA synthesis at 37 degrees C. Such interaction at 0 degrees C is very rapid, reaching 50% completion in about 15 s, and is not found with native NCS or when apo-NCS is added before the chromophore or when serum is included in the preincubation at 0 degrees C. Washing with apo-NCS or serum-containing (or-free) medium after preincubation of the cells with the chromophore at 0 degrees C fails to reverse the subsequenct inhibition of DNA synthesis.     

Isolation and disruption of the melanin pathway polyketide synthase gene of the softwood deep stain fungus Ceratocystis resinifera

Ceratocystis resinifera hyphae produce a black melanin pigment causing a deep stain in softwood logs. We exploited the homology of polyketide synthases to clone PKS1, a gene responsible for dihydroxynaphthalene-melanin biosynthesis in C. resinifera. Sequence analysis indicated that PKS1 has two introns near its 5(') end and encodes a 2188-amino acid polypeptide with five functional domains: beta-ketoacyl synthase, acyl transferase, two acyl carrier proteins and a thioesterase/Claisen cyclase. A gene disruption construct designed to replace a portion of PKS1 with a hygromycin resistance cassette was transformed into C. resinifera through Agrobacterium tumefaciens-mediated transformation. PKS1 null mutants had an albino phenotype, and pigmentation was restored by the addition of scytalone, a melanin pathway intermediate. The disruption of PKS1 and restoration of pigmentation with scytalone confirmed the presence of a dihydroxynaphthalene-melanin pathway in C. resinifera. The transformation method described in this paper is the first reported for a Ceratocystis species.     

NAH plasmid-mediated catabolism of anthracene and phenanthrene to naphthoic acids.

Pseudomonas fluorescens 5R contains an NAH7-like plasmid (pKA1), and P. fluorescens 5R mutant 5RL contains a bioluminescent reporter plasmid (pUTK21) which was constructed by transposon mutagenesis. Polymerase chain reaction mapping confirmed the localization of lux transposon Tn4431 300 bp downstream from the start of the nahG gene. Two degradation products, 2-hydroxy-3-naphthoic acid and 1-hydroxy-2-naphthoic acid, were recovered and identified from P. fluorescens 5RL as biochemical metabolites from the biotransformation of anthracene and phenanthrene, respectively. This is the first report which provides direct biochemical evidence that the naphthalene plasmid degradative enzyme system is involved in the degradation of higher-molecular-weight polycyclic aromatic hydrocarbons other than naphthalene.