Substrate specificity of naphthalene dioxygenase: effect of specific amino acids at the active site of the enzyme

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydrophobic, which is consistent with the enzyme's preference for aromatic hydrocarbon substrates. Although NDO catalyzes cis-dihydroxylation of a wide range of substrates, it is highly regio- and enantioselective. Site-directed mutagenesis was used to determine the contributions of several active-site residues to these aspects of catalysis. Amino acid substitutions at Asn-201, Phe-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no effect on product formation with naphthalene or biphenyl as substrates and had slight but significant effects on product formation from phenanthrene. Amino acid substitutions at Phe-352 resulted in the formation of cis-naphthalene dihydrodiol with altered stereochemistry [92 to 96% (+)-1R,2S], compared to the enantiomerically pure [>99% (+)-1R,2S] product formed by the wild-type enzyme. Substitutions at position 352 changed the site of oxidation of biphenyl and phenanthrene. Substitution of alanine for Asp-362, a ligand to the active-site iron, resulted in a completely inactive enzyme.     

Regioselectivity and enantioselectivity of naphthalene dioxygenase during arene cis-dihydroxylation: control by phenylalanine 352 in the alpha subunit

The naphthalene dioxygenase (NDO) system catalyzes the first step in the degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The enzyme has a broad substrate range and catalyzes several types of reactions including cis-dihydroxylation, monooxygenation, and desaturation. Substitution of valine or leucine at Phe-352 near the active site iron in the alpha subunit of NDO altered the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene and also changed the region of oxidation of biphenyl and phenanthrene. In this study, we replaced Phe-352 with glycine, alanine, isoleucine, threonine, tryptophan, and tyrosine and determined the activity with naphthalene, biphenyl, and phenanthrene as substrates. NDO variants F352W and F352Y were marginally active with all substrates tested. F352G and F352A had reduced but significant activity, and F352I, F352T, F352V, and F352L had nearly wild-type activities with respect to naphthalene oxidation. All active enzymes had altered regioselectivity with biphenyl and phenanthrene. In addition, the F352V and F352T variants formed the opposite enantiomer of biphenyl cis-3,4-dihydrodiol [77 and 60% (-)-(3S,4R), respectively] to that formed by wild-type NDO [>98% (+)-(3R,4S)]. The F352V mutant enzyme also formed the opposite enantiomer of phenanthrene cis-1,2-dihydrodiol from phenanthrene to that formed by biphenyl dioxygenase from Sphingomonas yanoikuyae B8/36. A recombinant Escherichia coli strain expressing the F352V variant of NDO and the enantioselective toluene cis-dihydrodiol dehydrogenase from Pseudomonas putida F1 was used to produce enantiomerically pure (-)-biphenyl cis-(3S,4R)-dihydrodiol and (-)-phenanthrene cis-(1S,2R)-dihydrodiol from biphenyl and phenanthrene, respectively.     

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. Received 25 September 2000/ Accepted in revised form 29 June 2001     

Metabolism of naphthalene, fluorene, and phenanthrene: preliminary characterization of a cloned gene cluster from Pseudomonas putida NCIB 9816.

A modified cloning procedure was used to obtain large DNA insertions (20 to 30 kb) from Pseudomonas putida NCIB 9816 that expressed polycyclic aromatic hydrocarbon (PAH) transformation activity in Escherichia coli HB101. Four subclones (16 [in both orientations], 12, and 8.5 kb in size) were constructed from the initial clones. Naphthalene, fluorene, and phenanthrene transformations were investigated in these eight NCIB 9816 clones by a simple agar plate assay method, which was developed to detect and identify potential PAH metabolites. Results indicated that the necessary genes encoding the initial ring fission of the three PAHs in E. coli cells are located in an 8.5-kb EcoRI-XhoI portion, but the lower-pathway genes are not present in a 38-kb neighborhood region. These NCIB 9816 clones could transform naphthalene and phenanthrene to salicylic acid and 1-hydroxy-2-naphthoic acid, respectively. With the same clones, fluorene was degraded to 9-hydroxyfluorene, 9-fluorenone, and two unidentified compounds. Genetic similarity between the NAH7 upper-pathway genes and the cloned NCIB 9816 genes was confirmed by Southern blot DNA-DNA hybridization. In spite of this genetic similarity, the abilities of the two clusters to transform multiple PAHs were different. Under our experimental conditions, only the metabolites from naphthalene transformation by the NAH7 clone (pE317) were detected, whereas the NCIB 9816 clones produced metabolites from all three PAHs.     

Naphthalene-dioxygenase-catalysed cis-dihydroxylation of azaarene derivatives: Part 1: 2-Pyridones and 2-quinolones

The scope of the biotransformation of 2-pyridone- and 2-quinolone-derived compounds by recombinant whole-cells of E. coli JM109(DE3)(pDTG141) expressing the naphthalene-dioxygenase system from Pseudomonas sp. NCIB 9816-4 was explored, using a series of N- and C-substituted derivatives. Among them, only the N-methyl substituted compounds were good substrates for a regio- and stereoselective dihydroxylation reaction leading to cis-dihydroxydihydro pyridone derivatives, corresponding to the general pattern expected for this enzyme. In the absence of dihydroxylation reactions, N-dealkylations and monohydroxylations on external methyl groups were observed.     

Stereoselective metabolism of anthracene and phenanthrene by the fungus Cunninghamella elegans.

The fungus Cunninghamella elegans oxidized anthracene and phenanthrene to form predominately trans-dihydrodiols. The metabolites were isolated by reversed-phase high-pressure liquid chromatography for structural and conformational analyses. Comparison of the circular dichroism spectrum of the fungal trans-1,2-dihydroxy-1,2-dihydroanthracene to that formed by rat liver microsomes indicated that the major enantiomer of the trans-1,2-dihydroxy-1,2-dihydroanthracene formed by C. elegans had an S,S absolute stereochemistry, which is opposite to the predominately 1R,2R dihydrodiol formed by rat liver microsomes. C. elegans oxidized phenanthrene primarily in the 1,2-positions to form trans-1,2-dihydroxy-1,2-dihydrophenanthrene. In addition, a minor amount of trans-3,4-dihydroxy-3,4-dihydrophenanthrene was detected. Metabolism at the K-region (9,10-positions) of phenanthrene was not detected. Comparison of the circular dichroism spectra of the phenanthrene trans-1,2- and trans-3,4-dihydrodiols formed by C. elegans to those formed by mammalian enzymes indicated that each of the dihydrodiols formed by C. elegans had an S,S absolute configuration. The results indicate that there are differences in both the regio- and stereoselective metabolism of anthracene and phenanthrene between the fungus C. elegans and rat liver microsomes.     

Oxidation of carbazole to 3-hydroxycarbazole by naphthalene 1,2-dioxygenase and biphenyl 2,3-dioxygenase

Abstract Naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4 and biphenyl dioxygenase from Beijerinckia sp. B8/36 oxidized the aromatic N-heterocycle carbazole to 3-hydroxycarbazole. Toluene dioxygenase from Pseudomonas putida F39/D did not oxidize carbazole. Transformations were carried out by mutant strains which oxidize naphthalene and biphenyl to cis -dihydrodiols, and with a recombinant E. coli strain expressing the structural genes of naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4. 3-Hydroxycarbazole is presumed to result from the dehydration of an unstable cis -dihydrodiol.     

Structural determinants of the substrate and stereochemical specificity of phosphotriesterase

Bacterial phosphotriesterase (PTE) catalyzes the hydrolysis of a wide variety of organophosphate nerve agents and insecticides. Previous kinetic studies with a series of enantiomeric organophosphate triesters have shown that the wild type PTE generally prefers the S(P)-enantiomer over the corresponding R(P)-enantiomers by factors ranging from 1 to 90. The three-dimensional crystal structure of PTE with a bound substrate analogue has led to the identification of three hydrophobic binding pockets. To delineate the factors that govern the reactivity and stereoselectivity of PTE, the dimensions of these three subsites have been systematically altered by site-directed mutagenesis of Cys-59, Gly-60, Ser-61, Ile-106, Trp-131, Phe-132, His-254, His-257, Leu-271, Leu-303, Phe-306, Ser-308, Tyr-309, and Met-317. These studies have shown that substitution of Gly-60 with an alanine within the small subsite dramatically decreased k(cat) and k(cat)/K(a) for the R(P)-enantiomers, but had little influence on the kinetic constants for the S(P)-enantiomers of the chiral substrates. As a result, the chiral preference for the S(P)-enantiomers was greatly enhanced. For example, the value of k(cat)/K(a) with the mutant G60A for the S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was 13000-fold greater than that for the corresponding R(P)-enantiomer. The mutation of I106, F132, or S308 to an alanine residue, which enlarges the small or leaving group subsites, caused a significant reduction in the enantiomeric preference for the S(P)-enantiomers, due to selective increases in the reaction rates for the R(P)-enantiomers. Enlargement of the large subsite by the construction of an H254A, H257A, L271A, or M317A mutant had a relatively small effect on k(cat)/K(a) for either the R(P)- or S(P)-enantiomers and thus had little effect on the overall stereoselectivity. These studies demonstrate that by modifying specific residues located within the active site of PTE, it is possible to dramatically alter the stereoselectivity and overall reactivity of the native enzyme toward chiral substrates.     

Selection for Growth on 3-Nitrotoluene by 2-Nitrotoluene-Utilizing Acidovorax sp. Strain JS42 Identifies Nitroarene Dioxygenases with Altered Specificities

Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3- or 4-nitrotoluene. Using both long- and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the α subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome.     

Rieske business: structure-function of Rieske non-heme oxygenases

Rieske non-heme iron oxygenases (RO) catalyze stereo- and regiospecific reactions. Recently, an explosion of structural information on this class of enzymes has occurred in the literature. ROs are two/three component systems: a reductase component that obtains electrons from NAD(P)H, often a Rieske ferredoxin component that shuttles the electrons and an oxygenase component that performs catalysis. The oxygenase component structures have all shown to be of the alpha3 or alpha3beta3 types. The transfer of electrons happens from the Rieske center to the mononuclear iron of the neighboring subunit via a conserved aspartate, which is shown to be involved in gating electron transport. Molecular oxygen has been shown to bind side-on in naphthalene dioxygenase and a concerted mechanism of oxygen activation and hydroxylation of the ring has been proposed. The orientation of binding of the substrate to the enzyme is hypothesized to control the substrate selectivity and regio-specificity of product formation.     

Degradation of phenanthrene and anthracene by Nocardia otitidiscaviarum strain TSH1, a moderately thermophilic bacterium

Aims:  The metabolism of phenanthrene and anthracene by a moderate thermophilic Nocardia otitidiscaviarum strain TSH1 was examined.
Methods and Results:  When strain TSH1 was grown in the presence of anthracene, four metabolites were identified as 1,2-dihydroxy-1,2-dihydroanthracene, 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, 2,3-dihydroxynaphthalene and benzoic acid using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC). Degradation studies with phenanthrene revealed 2,2'-diphenic acid, phthalic acid, 4-hydroxyphenylacetic acid, o -hydroxyphenylacetic acid, benzoic acid, a phenanthrene dihydrodiol, 4-[1-hydroxy(2-naphthyl)]-2-oxobut-3-enoic acid and 1-hydroxy-2-naphthoic acid (1H2NA), as detectable metabolites.
Conclusions:  Strain TSH1 initiates phenanthrene degradation via dioxygenation at the C-3 and C-4 or at C-9 and C-10 ring positions. Ortho -cleavage of the 9,10-diol leads to formation of 2,2'-diphenic acid. The 3,4-diol ring is cleaved to form 1H2NA which can subsequently be degraded through o -phthalic acid pathway. Benzoate does not fit in the previously published pathways from mesophiles. Anthracene metabolism seems to start with a dioxygenation at the 1 and 2 positions and ortho -cleavage of the resulting diol. The pathway proceeds probably through 2,3-dicarboxynaphthalene and 2,3-dihydroxynaphthalene. Degradation of 2,3-dihydroxynaphthalene to benzoate and transformation of the later to catechol is a possible route for the further degradation of anthracene.
Significance and Impact of the Study:  For the first time, metabolism of phenanthrene and anthracene in a thermophilic Nocardia strain was investigated.     

Efficient polycyclic aromatic hydrocarbons dihydroxylation in direct micellar systems

Optimization of whole-cell bioconversion of the polycyclic aromatic hydrocarbons (PAHs) anthracene, phenanthrene, and naphthalene to the enantiomerically pure corresponding cis-dihydroxydihydro derivatives by the Escherichia coli JM109 (pPS1778) recombinant strain, carrying the naphthalene dioxygenase and corresponding regulatory genes cloned from Pseudomonas fluorescens N3, in micellar systems, is presented. We show that direct microemulsion systems, where a nonionic surfactant such as 1.5% (v/v) Triton X-100 plus 0.6% to 1.0% (v/v) selected oils are able to solubilize the PAHs tested at relatively high concentrations (initial concentrations in the reaction medium > or =10 mM for naphthalene and phenanthrene and > or =2 mM for anthracene), and allow for more efficient substrate bioconversion. These media, while not affecting bacteria viability and performance, provide increased efficiency and final product yields (100% for naphthalene, >30% for anthracene, >60% for phenanthrene). The phase behavior of the direct microemulsion systems for the different substrates and oils utilized was monitored as a function of their volume fraction by light scattering experiments, and related to the bioconversion results. For anthracene and phenanthrene, the dihydroxylated products have an inhibitory effect on the conversion reactions, thus hindering complete turnover of the substrates. We ascertain that such inhibition is reversible because removal of the products formed allowed the process to start over at rates comparable to initial rates. To allow for complete conversion of the PAHs tested a stepwise or continuous separation of the product formed from the micellar reaction environment is being developed.     

Purification, characterization, and crystallization of the components of the nitrobenzene and 2-nitrotoluene dioxygenase enzyme systems

The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an alpha(3)beta(3) hexamer. The apparent K(m) of 2-nitrotoluene dioxygenase for 2NT was 20 muM, and that for naphthalene was 121 muM. The specificity constants were 7.0 muM(-1) min(-1) for 2NT and 1.2 muM(-1) min(-1) for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.     

Contrasting effects of a nonionic surfactant on the biotransformation of polycyclic aromatic hydrocarbons to cis-dihydrodiols by soil bacteria

The biotransformation of the polycyclic aromatic hydrocarbons (PAHs) naphthalene and phenanthrene was investigated by using two dioxygenase-expressing bacteria, Pseudomonas sp. strain 9816/11 and Sphingomonas yanoikuyae B8/36, under conditions which facilitate mass-transfer limited substrate oxidation. Both of these strains are mutants that accumulate cis-dihydrodiol metabolites under the reaction conditions used. The effects of the nonpolar solvent 2,2,4, 4,6,8,8-heptamethylnonane (HMN) and the nonionic surfactant Triton X-100 on the rate of accumulation of these metabolites were determined. HMN increased the rate of accumulation of metabolites for both microorganisms, with both substrates. The enhancement effect was most noticeable with phenanthrene, which has a lower aqueous solubility than naphthalene. Triton X-100 increased the rate of oxidation of the PAHs with strain 9816/11 with the effect being most noticeable when phenanthrene was used as a substrate. However, the surfactant inhibited the biotransformation of both naphthalene and phenanthrene with strain B8/36 under the same conditions. The observation that a nonionic surfactant could have such contrasting effects on PAH oxidation by different bacteria, which are known to be important for the degradation of these compounds in the environment, may explain why previous research on the application of the surfactants to PAH bioremediation has yielded inconclusive results. The surfactant inhibited growth of the wild-type strain S. yanoikuyae B1 on aromatic compounds but did not inhibit B8/36 dioxygenase enzyme activity in vitro.     

Proteomic distinction of renal oncocytomas and chromophobe renal cell carcinomas

Background

Renal oncocytomas (ROs) are benign epithelial tumors of the kidney whereas chromophobe renal cell carcinoma (chRCCs) are malignant renal tumors. The latter constitute 5–7% of renal neoplasias. ROs and chRCCs show pronounced molecular and histological similarities, which renders their differentiation demanding. We aimed for the differential proteome profiling of ROs and early-stage chRCCs in order to better understand distinguishing protein patterns.

Methods

We employed formalin-fixed, paraffin-embedded samples (six RO cases, six chRCC cases) together with isotopic triplex dimethylation and a pooled reference standard to enable cohort-wide quantitative comparison. For lysosomal-associated membrane protein 1 (LAMP1) and integrin alpha-V (ITGAV) we performed corroborative immunohistochemistry (IHC) in an extended cohort of 42 RO cases and 31 chRCC cases.

Results

At 1% false discovery rate, we identified?>?3900 proteins, of which?>?2400 proteins were consistently quantified in at least four RO and four chRCC cases. The proteomic expression profiling discriminated ROs and chRCCs and highlighted established features such as accumulation of mitochondrial proteins in ROs together with emphasizing the accumulation of endo-lysosomal proteins in chRCCs. In line with the proteomic data, IHC showed enrichment of LAMP1 in chRCC and of ITGAV in RO.

Conclusion

We present one of the first differential proteome profiling studies on ROs and chRCCs and highlight differential abundance of LAMP1 and ITGAV in these renal tumors.

     

Crystal structure of human cytochrome P450 2D6

Cytochrome P450 2D6 is a heme-containing enzyme that is responsible for the metabolism of at least 20% of known drugs. Substrates of 2D6 typically contain a basic nitrogen and a planar aromatic ring. The crystal structure of human 2D6 has been solved and refined to 3.0A resolution. The structure shows the characteristic P450 fold as seen in other members of the family, with the lengths and orientations of the individual secondary structural elements being very similar to those seen in 2C9. There are, however, several important differences, the most notable involving the F helix, the F-G loop, the B'helix, beta sheet 4, and part of beta sheet 1, all of which are situated on the distal face of the protein. The 2D6 structure has a well defined active site cavity above the heme group, containing many important residues that have been implicated in substrate recognition and binding, including Asp-301, Glu-216, Phe-483, and Phe-120. The crystal structure helps to explain how Asp-301, Glu-216, and Phe-483 can act as substrate binding residues and suggests that the role of Phe-120 is to control the orientation of the aromatic ring found in most substrates with respect to the heme. The structure has been compared with published homology models and has been used to explain much of the reported site-directed mutagenesis data and help understand the metabolism of several compounds.     

Enantiomeric Composition of the trans-Dihydrodiols Produced from Phenanthrene by Fungi

The trans-dihydrodiols produced during the metabolism of phenanthrene by Cunninghamella elegans, Syncephalastrum racemosum, and Phanerochaete chrysosporium were purified by high-performance liquid chromatography (HPLC). The enantiomeric compositions and optical purities of the trans-dihydrodiols were determined to compare interspecific differences in the regio- and stereoselectivity of the fungal enzymes. Circular dichroism spectra of the trans-dihydrodiols were obtained, and the enantiomeric composition of each preparation was analyzed by HPLC with a chiral stationary-phase column. The phenanthrene trans-1,2-dihydrodiol produced by C. elegans was a mixture of the 1R,2R and 1S,2S enantiomers in variable proportions. The phenanthrene trans-3,4-dihydrodiol produced by P. chrysosporium was the optically pure 3R,4R enantiomer, but that produced by S. racemosum was a 68:32 mixture of the 3R,4R and 3S,4S enantiomers. The phenanthrene trans-9,10-dihydrodiol produced by P. chrysosporium was predominantly the 9S,10S enantiomer, but those produced by C. elegans and S. racemosum were predominantly the 9R,10R enantiomer. The results indicate that although different fungi may exhibit similar regioselectivity, there still may be differences in stereoselectivity that depend on the species and the cultural conditions.     

Determination of the substrate binding mode to the active site iron of (S)-2-hydroxypropylphosphonic acid epoxidase using 17O-enriched substrates and substrate analogues

(S)-2-Hydroxypropylphosphonic acid epoxidase (HppE) is an O2-dependent, nonheme Fe(II)-containing oxidase that converts (S)-2-hydroxypropylphosphonic acid ((S)-HPP) to the regio- and enantiomerically specific epoxide, fosfomycin. Use of (R)-2-hydroxypropylphosphonic acid ((R)-HPP) yields the 2-keto-adduct rather than the epoxide. Here we report the chemical synthesis of a range of HPP analogues designed to probe the basis for this specificity. In past studies, NO has been used as an O2 surrogate to provide an EPR probe of the Fe(II) environment. These studies suggest that O2 binds to the iron, and substrates bind in a single orientation that strongly perturbs the iron environment. Recently, the X-ray crystal structure showed direct binding of the substrate to the iron, but both monodentate (via the phosphonate) and chelated (via the hydroxyl and phosphonate) orientations were observed. In the current study, hyperfine broadening of the homogeneous S = 3/2 EPR spectrum of the HppE-NO-HPP complex was observed when either the hydroxyl or the phosphonate group of HPP was enriched with 17O (I = 5/2). These results indicate that both functional groups of HPP bind to Fe(II) ion at the same time as NO, suggesting that the chelated substrate binding mode dominates in solution. (R)- and (S)-analogue compounds that maintained the core structure of HPP but added bulky terminal groups were turned over to give products analogous to those from (R)- and (S)-HPP, respectively. In contrast, substrate analogues lacking either the phosphonate or hydroxyl group were not turned over. Elongation of the carbon chain between the hydroxyl and phosphonate allowed binding to the iron in a variety of orientations to give keto and diol products at positions determined by the hydroxyl substituent, but no stable epoxide was formed. These studies show the importance of the Fe(II)-substrate chelate structure to active antibiotic formation. This fixed orientation may align the substrate next to the iron-bound activated oxygen species thought to mediate hydrogen atom abstraction from the nearest substrate carbon.     

Cytokeratin 20 immunoreactivity in renal oncocytomas.

Cytokeratins (CKs) are a group of 20 antigenically distinct intermediate filaments, generally confined to epithelia and their neoplasms. Immunostaining for CKs, in particular coordinate staining for CK7 and CK20, has become a useful tool in diagnostic pathology. Although studies defining CK distribution in neoplasms identify 0--7.7% of renal cell carcinomas (RCCs) positive for CK20, none has described the incidence of CK20 immunopositivity in renal oncocytomas (ROs). Distinction between RCC and RO may be difficult but this distinction is clinically significant, prompting us to establish the incidence of CK20 positivity in RO. We selected fifteen surgical cases of RO from our archives and studied their immunoreactivity for CKs including CK7 and CK20; 12/15 (80%) were positive for CK20, with variation in the number of cells staining. There was also variation in the distribution of CKs within the cells, including diffuse cytoplasmic, perinuclear, and a punctate or dot-like pattern. Such punctate staining corresponds to cytoplasmic balls of intermediate filaments and has been described with CAM 5.2 in RO and CK20 in Merkel cell carcinomas. Our findings suggest that CK20 immunohistochemistry is a useful tool for distinguishing RCCs from ROs. (J Histochem Cytochem 49:919-920, 2001)     

Regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

The regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene was examined with mutant and recombinant strains expressing naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. The initial oxidation products were isolated and identified by gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry. Salicylate-induced cells of Pseudomonas sp. strain 9816/11 and isopropyl-beta-D-thiogalactopyranoside-induced cells of Escherichia coli JM109(DE3)(pDTG141) oxidized fluorene to (+)-(3S,4R)-cis-3,4-dihydroxy-3,4-dihydrofluorene (80 to 90% relative yield; > 95% enantiomeric excess [ee]) and 9-fluorenol (< 10% yield). The same cells oxidized dibenzofuran to (1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzofuran (60 to 70% yield; > 95% ee) and (3S,4R)-cis-3, 4-dihydroxy-3,4-dihydrodibenzofuran (30 to 40% yield; > 95% ee). Induced cells of both strains, as well as the purified dioxygenase, also oxidized dibenzothiophene to (+)-(1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzothiophene (84 to 87% yield; > 95% ee) and dibenzothiophene sulfoxide (< 15% yield). The major reaction catalyzed by naphthalene dioxygenase with each substrate was stereospecific dihydroxylation in which the cis-dihydrodiols were of identical regiochemistry and of R configuration at the benzylic center adjacent to the bridgehead carbon atom. The regiospecific oxidation of dibenzofuran differed from that of the other substrates in that a significant amount of the minor cis-3,4-dihydrodiol regioisomer was formed. The results indicate that although the absolute stereochemistry of the cis-diene diols was the same, the nature of the bridging atom or heteroatom influenced the regiospecificity of the reactions catalyzed by naphthalene dioxygenase.