Evaluación de test rápidos y diseño de una estrategia para la detección y caracterización de carbapenemasas en cepas de bacilos gramnegativos / Test evaluation and strategy proposal to detect and to characterize carbapenemase-producing gram negative bacilli

Resumen Introducción: La detección de bacilos gramnegativos productores de carbapenemasas es compleja, existiendo actualmente varios test disponibles. La confirmación mediante la caracterización molecular de la enzima no está disponible en todos los laboratorios del país. Objetivo: Plantear una estrategia rápida, eficiente y sencilla para la detección y confirmación de carbapenemasas en cepas de bacilos gramnegativos. Material y Métodos: Se utilizaron 39 aislados productores y ocho no productores de carbapenemasas para evaluar los test fenotípicos Carba NP, CarbAcineto NP, Blue-Carba y validar el test molecular Xpert® Carba-R directo de la colonia en comparación con RPC convencional. Resultados: La sensibilidad para Carba NP, CarbAcineto NP y Blue-Carba fue de 79,5; 87,2 y 84,6%, respectivamente; mientras que la especificidad fue de 100; 100 y 87,5%, respectivamente. La concordancia entre RPC convencional y Xpert® Carba-R fue de 100%. El límite de detección para Xpert® Carba-R fue diferente según el tipo de carbapenemasa: 40,8 ufc/reacción par KPC y NDM y 30,6 ufc/reacción para VIM. Discusión: En aislados con susceptibilidad disminuida a carbapenémicos se propone realizar un tamizaje con CarbAcineto NP, para luego caracterizar la carbapenemasa con Xpert® Carba-R y adoptar las medidas de contención específica: para cada caso.

Introduction: The detection of carbapenemase-producing gram negative bacilli is complicated, because there are available multiple options of test. The confirmation of the enzyme by molecular characterization is not available in all laboratories in our country. Objective: To propose a fast, efficient and simple strategy to detect and confirm CPB. Materials and Methods: 39 CPB isolates and 8 non-producing were used to evaluate the phenotypic test Carba NP, CarbAcineto NP and Blue-Carba, validating the test Xpert® Carba-R, to be used directly with bacterial colonies with conventional PCR. Results: The sensitivity of Carba NP, CarbAcineto NP and Blue-Carba was 79,5; 87,2 y 84,6%, respectively; and specificity was 79.5; 87.2 and 84.6%, respectively. The limit of detection of Xpert® Carba-R was different for each carbapenemasa: 40.8 ufc/reaction to KPC and NDM and 30.6 ufc/reaction to VIM. Discussion: On isolates with decreased susceptibility to carbapenems we propose to use as screening the test CarbAcineto NP, follow by Xpert®Carba-R to characterize the carbapenemase and adopt specific infection control measures.

Factores asociados a infecciones urinarias producidas por enterobacterias productoras de β -lactamasas de espectro extendido: una cohorte prospectiva / Factors associated with extended-spectrum betalactamases-producing organisms among patients with urinary tract infections: a prospective cohort study

Background: Urinary tract infections (UTIs) caused by extended-spectrum betalactamases (ESBL) are an increasingly common problem. Aim: To develop an association model to allow an early detection of ESBL-producing microorganisms. Methods: A prospective observational cohort study was undertaken among patients admitted with a diagnosis of culture-proven UTI to the Internal Medicine Ward of the Hospital Naval Almirante Nef between February and November, 2011. Patients with polimicrobial cultures were excluded from analyses, which was undertaken using multiple logistic regression. Results: Two-hundred and forty-nine patients were analysed and 35 (14%) presented an ESBL-producing microorganism. Seventy-one percent were female and the mean age was 70,7 ± 16,9 years. A history of a recent hospitalization (< 3 months) or institutionalization (p = 0.027), previous infections by an ESBL-producing bacteria (p < 0.001), recent antimicrobial use (p = 0.013) and metastatic cancer (p = 0.007) were independently associated with a current UTI with an ESBL-producing pathogen. Discussion: Our findings are similar to those found in other populations. This tool offers assistance to clinicians who need to choose an appropriate antimicrobial therapy. This model needs to be validated prior to implementation.

Introducción: La infección del tracto urinario (ITU) por microorganismos productores de β-lactamasas de espectro extendido (BLEE) es un problema infectológico creciente. Objetivo: Determinar factores de riesgo predisponentes a infecciones por microorganismos productores de BLEE. Pacientes y Método: Cohorte prospectiva de pacientes > 18 años ingresados al Servicio de Medicina Interna del Hospital Naval Almirante Nef de Viña del Mar desde febrero a noviembre de 2011 con diagnóstico de ITU confirmado en un urocultivo. Se excluyeron pacientes con urocultivos polimicrobianos. El análisis se hizo mediante una regresión logística múltiple. Resultados: Se analizaron 249 pacientes, 35 (14%) presentaron un microorganismo productor de BLEE. El 71% fueron mujeres y la edad promedio 70,7 ± 16,9 años. El antecedente de hospitalización en los últimos tres meses o el vivir institucionalizado (p = 0,027), la infección por bacteria productora de BLEE previa (p < 0,001), el uso de antimicrobianos recientes (p = 0,013) y el antecedente de cáncer metastásico (p = 0,007) se asociaron a la producción de BLEE. Discusión: Los factores encontrados en la presente cohorte están de acuerdo a lo descrito en otras poblaciones. Esta herramienta ofrece asistencia para el médico clínico en la selección de la antibioterapia más apropiada. Es necesario validar este modelo previo a su implementación.

Gene cluster of Rhodothermus marinus high-potential iron-sulfur Protein: oxygen oxidoreductase, a caa(3)-type oxidase belonging to the superfamily of heme-copper oxidases.

The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains an oxygen reductase, which uses HiPIP (high potential iron-sulfur protein) as an electron donor. The structural genes encoding the four subunits of this HiPIP:oxygen oxidoreductase were cloned and sequenced. The genes for subunits II, I, III, and IV (named rcoxA to rcoxD) are found in this order and seemed to be organized in an operon of at least five genes with a terminator structure a few nucleotides downstream of rcoxD. Examination of the amino acid sequence of the Rcox subunits shows that the subunits of the R. marinus enzyme have homology to the corresponding subunits of oxidases belonging to the superfamily of heme-copper oxidases. RcoxB has the conserved histidines involved in binding the binuclear center and the low-spin heme. All of the residues proposed to be involved in proton transfer channels are conserved, with the exception of the key glutamate residue of the D-channel (E(278), Paracoccus denitrificans numbering). Analysis of the homology-derived structural model of subunit I shows that the phenol group of a tyrosine (Y) residue and the hydroxyl group of the following serine (S) may functionally substitute the glutamate carboxyl in proton transfer. RcoxA has an additional sequence for heme C binding, after the Cu(A) domain, that is characteristic of caa(3) oxidases belonging to the superfamily. Homology modeling of the structure of this cytochrome domain of subunit II shows no marked electrostatic character, especially around the heme edge region, suggesting that the interaction with a redox partner is not of an electrostatic nature. This observation is analyzed in relation to the electron donor for this caa(3) oxidase, the HiPIP. In conclusion, it is shown that an oxidase, which uses an iron-sulfur protein as an electron donor, is structurally related to the caa(3) class of heme-copper cytochrome c oxidases. The data are discussed in the framework of the evolution of oxidases within the superfamily of heme-copper oxidases.

Hydrogenophaga intermedia sp. nov., a 4-aminobenzenesulfonate degrading organism.

The taxonomic status of a gram-negative, oxidase positive rod (strain S1) able to degrade 4-aminobenzenesulfonate was studied using a polyphasic approach. Chemotaxonomic investigations of quinones and polar lipids established the allocation of this strain to the beta-subclass of the Proteobacteria and revealed similarities to Hydrogenophaga palleronii. 16S rRNA sequence comparisons demonstrated that this strain clusters phylogenetically with H. palleronii and H. taeniospiralis, but clearly represents a new species. The fatty acid patterns and substrate utilization profile displayed similarity to the characteristics of the four validly published species of Hydrogenophaga, although clear differentiating characters were also observed. No close similarities between the type strains of H. palleronii and H. taeniospiralis were detected in hybridization experiments with the genomic DNAs. On basis of these results, the new species Hydrogenophaga intermedia sp. nov. is proposed, with the type strain S1T (= DSM 5680).

Properties of the methylcobalamin:H4folate methyltransferase involved in chloromethane utilization by Methylobacterium sp. strain CM4.

Methylobacterium sp. strain CM4 is a strictly aerobic methylotrophic proteobacterium growing with chloromethane as the sole carbon and energy source. Genetic evidence and measurements of enzyme activity in cell-free extracts have suggested a multistep pathway for the conversion of chloromethane to formate. The postulated pathway is initiated by a corrinoid-dependent methyltransferase system involving methyltransferase I (CmuA) and methyltransferase II (CmuB), which transfer the methyl group of chloromethane onto tetrahydrofolate (H4folate) [Vannelli et al. (1999) Proc. Natl Acad. Sci. USA 96, 4615-4620]. We report the overexpression in Escherichia coli and the purification to apparent homogeneity of methyltransferase II. This homodimeric enzyme, with a subunit molecular mass of 33 kDa, catalyzed the conversion of methylcobalamin and H4folate to cob(I)alamin and methyl-H4folate with a specific activity of 22 nmol x min-1 x (mg protein)-1. The apparent kinetic constants for H4folate were: Km = 240 microM, Vmax = 28.5 nmol x min-1 x (mg protein)-1. The reaction appeared to be first order with respect to methylcobalamin at concentrations up to 2 mM, presumably reflecting the fact that methylcobalamin is an artificial substitute for the methylated methyltransferase I, the natural substrate of the enzyme. Tetrahydromethanopterin, a coenzyme also present in Methylobacterium, did not serve as a methyl group acceptor for methyltransferase II. Purified methyltransferase II restored chloromethane dehalogenation by a cell free extract of a strain CM4 mutant defective in methyltransferase II.

Two intertwined methylation activities of the MmeI restriction-modification class-IIS system from Methylophilus methylotrophus.

The class-IIS restriction endonuclease, R.MmeI, was isolated from Methylophilus methylotrophus. It was originally described as a monomeric enzyme, with the native Mr 105000+/-7000, which did not cleave DNA efficiently [Boyd et al. (1986) Nucleic Acids Res. 14, 5255-5274; Tucholski et al. (1995) Gene 157, 87-92]. However, it was discovered that R.MmeI endonucleolytic activity is enhanced by S-adenosyl-l-methionine (AdoMet) and sinefungin, an analogue of AdoMet. Surprisingly, the purified R.MmeI endonuclease was found to have a second enzymatic activity, namely methylation of the adenine residue to N6-methyladenine in the top strand of the MmeI-recognition sequence, 5'-TCCR*AC-3' (*A=meA. The R.MmeI methylating activity requires AdoMet and is increased in the presence of several divalent cations, 20-fold by Mg2+ or Ca2+, and less by Mn2+, Zn2+ and Co2+; however, methylation is inhibited entirely by sinefungin, at concentrations above 9microM. The latter observation shows that the enhancing effect of AdoMet or sinefungin on the DNA cleavage was not related to the process of DNA methylation. Furthermore, a second component of the MmeI restriction-modification system, a M.MmeI methyltransferase, was isolated and purified. The M.MmeI protein was found to have an Mr of 48000+/-2000 (under denaturing conditions) and to methylate both adenine residues (*A) in the MmeI-recognition sequence 5'-TCCR*AC-3'/3'-*AGGYTG-5'. Methylation of the top strand does not inhibit the DNA cleavage by R.MmeI, whereas methylation of both DNA strands blocks the cleavage process.

Biochemical and genetic characterization of a gentisate 1, 2-dioxygenase from Sphingomonas sp. strain RW5.

A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC 1.13.11.4), gtdA, from Sphingomonas sp. strain RW5 was cloned and sequenced. The gtdA gene encodes a 350-amino-acid polypeptide with a predicted size of 38.85 kDa. Comparison of the gtdA gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no significant homology except for a low similarity (27%) to the 1-hydroxy-2-naphthoate dioxygenase (phdI) of the phenanthrene degradation in Nocardioides sp. strain KP7 (T. Iwabuchi and S. Harayama, J. Bacteriol. 179:6488-6494, 1997). This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases. The gene was subcloned and hyperexpressed in E. coli. The resulting product was purified to homogeneity and partially characterized. Under denaturing conditions, the polypeptide exhibited an approximate size of 38.5 kDa and migrated on gel filtration as a species with a molecular mass of 177 kDa. The enzyme thus appears to be a homotetrameric protein. The purified enzyme stoichiometrically converted gentisate to maleylpyruvate, which was identified by gas chromatography-mass spectrometry analysis as its methyl ester. Values of affinity constants (Km) and specificity constants (Kcat/Km) of the enzyme were determined to be 15 microM and 511 s-1 M-1 x 10(4) for gentisate and 754 microM and 20 s-1 M-1 x 10(4) for 3, 6-dichlorogentisate. Three further open reading frames (ORFs) were found downstream of gtdA. The deduced amino acid sequence of ORF 2 showed homology to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomerase superfamily and glutathione reductase superfamily, respectively.

The alkene monooxygenase from Xanthobacter Py2 is a binuclear non-haem iron protein closely related to toluene 4-monooxygenase.

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter Py2 have been sequenced. The predicted amino acid sequence of the first ORF shows homology with the iron binding subunits of binuclear non-haem iron containing monooxygenases including benzene monooxygenase, toluene 4-monooxygenase (> 60% sequence similarity) and methane monooxygenase (> 40% sequence similarity) and that the necessary sequence motifs associated with iron co-ordination are also present. Secondary structure prediction based on the amino acid sequence showed that the predominantly alpha-helical structure that surrounds the binuclear iron binding site was conserved allowing the sequence to be modelled on the co-ordinates of the methane monooxygenase alpha-subunit. Significant differences in the residues forming the hydrophobic cavity which forms the substrate binding site are discussed with reference to the differences in reaction specificity and stereospecificity of binuclear non-haem iron monooxygenases.

Molecular cloning and expression of fatty acid alpha-hydroxylase from Sphingomonas paucimobilis.

Fatty acid alpha-hydroxylase (FAAH) catalyzes the initial reaction in alpha-oxidation of fatty acid to produce 2-hydroxy fatty acid. FAAH activity has been detected in a wide range of organisms from prokaryotes to eukaryotes. Here, we describe cloning of the FAAH gene from Sphingomonas paucimobilis, a sphingolipid- and 2-hydroxymyristic acid-rich bacterium. The isolated gene encoded 415 amino acids. A homology search revealed that amino acid sequences highly conserved in cytochrome P450 (P450) were present in FAAH. Although the heme-binding cysteine was recognizable at position 361, the consensus in the heme-binding region was modified by an insertion. Overall, FAAH has no significant identity to the known P450s. CO difference spectrum of recombinant FAAH showed the characteristic one of P450, except this peak was at 445 nm. These results suggest bacterial FAAH is a novel member of the P450 superfamily.

Non-enzymatic and enzymatic hydrolysis of alkyl halides: a haloalkane dehalogenation enzyme evolved to stabilize the gas-phase transition state of an SN2 displacement reaction.

The semiempirical PM3 method, calibrated against ab initio HF/6-31+G(d) theory, has been used to elucidate the reaction of 1, 2-dichloroethane (DCE) with the carboxylate of Asp-124 at the active site of haloalkane dehalogenase of Xanthobacter autothropicus. Asp-124 and 13 other amino acid side chains that make up the active site cavity (Glu-56, Trp-125, Phe-128, Phe-172, Trp-175, Leu-179, Val-219, Phe-222, Pro-223, Val-226, Leu-262, Leu-263, and His-289) were included in the calculations. The three most significant observations of the present study are that: (i) the DCE substrate and Asp-124 carboxylate, in the reactive ES complex, are present as an ion-molecule complex with a structure similar to that seen in the gas-phase reaction of AcO- with DCE; (ii) the structures of the transition states in the gas-phase and enzymatic reaction are much the same where the structure formed at the active site is somewhat exploded; and (iii) the enthalpies in going from ground states to transition states in the enzymatic and gas-phase reactions differ by only a couple kcal/mol. The dehalogenase derives its catalytic power from: (i) bringing the electrophile and nucleophile together in a low-dielectric environment in an orientation that allows the reaction to occur without much structural reorganization; (ii) desolvation; and (iii) stabilizing the leaving chloride anion by Trp-125 and Trp-175 through hydrogen bonding.

Purification and characterization of acetone carboxylase from Xanthobacter strain Py2.

Acetone metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by a carboxylation reaction forming acetoacetate as the first detectable product. In this study, acetone carboxylase, the enzyme catalyzing this reaction, has been purified to homogeneity and characterized. Acetone carboxylase was comprised of three polypeptides with molecular weights of 85,300, 78,300, and 19,600 arranged in an alpha2beta2gamma2 quaternary structure. The carboxylation of acetone was coupled to the hydrolysis of ATP and formation of 1 mol AMP and 2 mol inorganic phosphate per mol acetoacetate formed. ADP was also formed during the course of acetone consumption, but only accumulated at low, substoichiometric levels ( approximately 10% yield) relative to acetoacetate. Inorganic pyrophosphate could not be detected as an intermediate or product of acetone carboxylation. In the absence of CO2, acetone carboxylase catalyzed the acetone-dependent hydrolysis of ATP to form both ADP and AMP, with ADP accumulating to higher levels than AMP during the course of the assays. Acetone carboxylase did not have inorganic pyrophosphatase activity. Acetone carboxylase exhibited a Vmax for acetone carboxylation of 0.225 micromol acetoacetate formed min-1.mg-1 at 30 degrees C and pH 7.6 and apparent Km values of 7.80 microM (acetone), 122 microM (ATP), and 4. 17 mM (CO2 plus bicarbonate). These studies reveal molecular properties of the first bacterial acetone-metabolizing enzyme to be isolated and suggest a novel mechanism of acetone carboxylation coupled to ATP hydrolysis and AMP and inorganic phosphate formation.

Identification and mutation of a gene required for glycerate kinase activity from a facultative methylotroph, Methylobacterium extorquens AM1.

A gene (gckA) responsible for the activity of glycerate kinase has been identified within a chromosomal fragment of the serine cycle methylotroph Methylobacterium extorquens AM1. A mutation in gckA leads to a specific C1-negative phenotype. The polypeptide sequence derived from gckA showed high similarity to a product of ttuD essential for tartrate metabolism in Agrobacterium vitis. Our data suggest that gckA and ttuD might be structural genes for glycerate kinase and that the serine cycle and the tartrate utilization pathway share a series of reactions.

Highly efficient control of iron-containing nitrile hydratases by stoichiometric amounts of nitric oxide and light.

The reaction of two iron-containing nitrile hydratases (NHase) with NO has been studied: NHase from Rhodococcus sp. R312, which is probably similar to the photosensitive N771 NHase, and the new NHase from Comamonas testosteroni NI1 whose aminoacid sequence is quite different from those of BR312 and N771 NHases. Both enzymes are equally inactivated after addition of stoichiometric amounts of NO added as an anaerobic solution or produced in situ under physiological conditions by a rat brain NO-synthase. Both enzymes are reactivated by photoirradiation, and two cycles of NO inactivation/photoactivation can be performed without significant loss of activity. Both iron-containing NHases have a high affinity for NO, similar to that of methemoglobin.

Organization of methylamine utilization genes (mau) in 'Methylobacillus flagellatum ' KT and analysis of mau mutants.

The organization of genes involved in utilization of methylamine (mau genes) was studied in the obligate methylotroph 'Methylobacillus flagellatum' KT. Nine open reading frames were identified as corresponding to the genes mauFBEDAGLMN. In addition, an open reading frame (orf-1 encoding a polypeptide with unknown function was identified upstream of the mau gene cluster. Subclones of the 'M. flagellatum' KT gene cluster were used for complementation of a series of chemically induced mau mutants of 'M. flagellatum' KT. Mutants in mauF, mauB, mauE/D, mauA, mauG, mauL and mauM were identified. Two mutants (mau-18 and mau-19) were not complemented by the known mau genes. Since none of the chemically induced mutants studies had a defect of orf-1 or mauN, inserting mutants in these genes were constructed. Phenotypically the mutants fell into three groups. The mauF, mauB, mauE/D, mauA, mauG, mauL and mauM mutants do not grow on methylamine as a source of carbon and lack methylamine dehydrogenase activity, but they synthesize both the large and the small subunit polypeptides albeit at different ratios. The mau-18 and mau-19 mutants do not grow on methylamine as a source of carbon, and lack both methylamine dehydrogenase activity and the methylamine dehydrogenase subunits. The orf-1 and mauN mutants grow on methylamine as a source of carbon and synthesize wild-type levels of methylamine dehydrogenase. It has been shown earlier that the product of the mauM gene is not required for synthesis of active methylamine dehydrogenase in Methylobacterium extorquens AM1 and Paracoccus denitrificans. However, MauM is required for synthesis of functional methylamine dehydrogenase in 'M. flagellatum'.

Benzoyl-CoA reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism. A study of adenosinetriphosphatase activity, ATP stoichiometry of the reaction and EPR properties of the enzyme.

An enzyme was recently described, benzoyl-CoA reductase (dearomatizing), which catalyses the ATP-driven reduction of the aromatic ring of benzoyl-CoA yielding a non-aromatic CoA thioester, ADP and phosphate [Boll, M. & Fuchs, G. (1995) Eur. J. Biochem. 234, 921-933]. The 170-kDa enzyme consists of four different subunits and contains approximately 12 Fe and acid-labile sulfur/mol. Benzoyl-CoA reductase exhibits ATPase activity in the absence of substrate. It is shown that only the reduced form of this iron-sulfur protein has ATPase activity. ATPase activity is reversibly lost when the enzyme is oxidized by thionine; reduction of the enzyme fully restores ATPase and ring-reduction activity. 2 mol ATP are hydrolyzed/2 mol electrons transferred in the course of the reaction. The product ADP acts as competitive inhibitor (Ki = 1.1 mM) for ATP in benzoyl-CoA reduction; ADP inhibits ATPase activity to the same extent as ring-reduction activity. EPR investigation of the dithionite-reduced enzyme suggested the presence of two separate [2Fe-2S] clusters and two interacting [4Fe-4S] clusters. Addition of MgATP to the reduced enzyme resulted in a new isotropic signal at g = 5.15 and a weak signal at g = 12; in controls with MgADP only a minor signal at g = 5.15 was observed. The positions, shapes and temperature dependencies of these MgATP-induced signals are indicative for excited states of a S = 7/2 spin multiplet. The [2Fe-2S] signals were not affected by ATP, but one of the [4Fe-4S] clusters became slowly oxidized. Addition of both benzoyl-CoA and MgATP resulted in a major oxidation of the iron-sulfur clusters accompanied by the appearance of some minor signals of unknown origin in the g = 2.037-1.96 region. Neither the benzoyl-CoA plus MgATP-oxidized nor the thionine-oxidized enzyme showed the ATP-dependent formation of the high-spin signals of the reduced enzyme. At present we hypothesize that the S = 7/2 signal is due to an ATP-induced change of one of the [4Fe-4S] clusters. The data suggest that hydrolysis of MgATP is required to activate the enzyme; in the absence of substrate the energy involved in this activation dissipates. MgATP-driven formation of this excited state of the reduced enzyme rather than transfer of electrons from the reduced enzyme to the aromatic substrate appears to be the rate-limiting step in the catalytic cycle. We suggest that the excited state is required to overcome the high activation energy associated with the loss of the aromatic character and/or to render ring reduction irreversible.

Purification and molecular characterization of the electron transfer protein of methanesulfonic acid monooxygenase.

A novel serine pathway methylotroph, strain M2, capable of utilizing methanesulfonic acid (MSA) as a sole source of carbon and energy was investigated. The initial step in the biodegradative pathway of MSA in strain M2 involved an inducible NADH-specific monooxygenase enzyme (MSAMO). Fractionation of MSAMO active cell extracts by ion-exchange chromatography led to the loss of MSAMO activity. Activity was restored by mixing three distinct protein fractions, designated A, B, and C. Further purification to homogeneity of component C indicated that the polypeptide was acidic, with a pI of 3.9, and contained an iron-sulfur center with spectral characteristics similar to those of other proteins containing Rieske [2Fe-2S] centers. The size of the protein subunit and the similarity of the N-terminal sequence to those of ferredoxin components of other oxygenase enzymes have suggested that component C is a specific electron transfer protein of the MSAMO which contains a Rieske [2Fe-2S] cluster. The gene encoding component C of MSAMO was cloned and sequenced, and the predicted protein sequence was compared with those of other Rieske [2Fe-2S]-center-containing ferredoxins. MSAMO appears to be a novel combination of oxygenase elements in which an enzyme related to aromatic-ring dioxygenases attacks a one-carbon (C1) compound via monooxygenation.

Purification to homogeneity and reconstitution of the individual components of the epoxide carboxylase multiprotein enzyme complex from Xanthobacter strain Py2.

Epoxide metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by an NADPH- and NAD+-dependent carboxylation reaction that forms beta-keto acids as products. Epoxide carboxylase, the enzyme catalyzing this reaction, was resolved from the soluble fraction of cell-free extracts into four protein components that are obligately required for functional reconstitution of epoxide carboxylase activity. One of these components, component II, has previously been purified and characterized as an NADPH:disulfide oxidoreductase. In the present study, the three additional epoxide carboxylase components have been purified to homogeneity and characterized. These component proteins are as follows: component I, a homohexameric protein consisting of 41.7-kDa subunits; component III, a dimeric protein consisting of 26.0- and 26.2-kDa polypeptides; and component IV, a dimeric protein consisting of a single 25.4-kDa polypeptide. Component I contained 5 mol of tightly bound zinc per mol of protein. Component I was specifically inactivated by methylepoxypropane, a time-dependent irreversible inactivator of epoxide carboxylase activity, suggesting that this component plays an integral role in epoxide binding and activation. No metals or organic cofactors were detected for components III and IV. The molecular weights, N-terminal sequences, and amino acid compositions of the purified epoxide carboxylase components were determined and found to correlate with open reading frames within and adjacent to a cloned fragment of DNA that complements Xanthobacter Py2 mutants defective in epoxide degradation. Using the purified epoxide carboxylase system, epoxide carboxylation was found to be stoichiometrically coupled to the transhydrogenation of pyridine nucleotide cofactors according to the following equation: epoxypropane + CO2 + NADPH + NAD+ --> acetoacetate + H+ + NADP+ + NADH.

A novel type of pyridine nucleotide-disulfide oxidoreductase is essential for NAD+- and NADPH-dependent degradation of epoxyalkanes by Xanthobacter strain Py2.

Epoxide degradation in cell extracts of Xanthobacter strain Py2 has been reported to be dependent on NAD+ and dithiols. This multicomponent system has now been fractionated. A key protein encoded by a DNA fragment complementing a Xanthobacter strain Py2 mutant unable to degrade epoxides was purified and analyzed. This NADP-dependent protein, a novel type of pyridine nucleotide-disulfide oxidoreductase, is essential for epoxide degradation. NADPH, acting as the physiological cofactor, replaced the dithiols in epoxide conversion.

Involvement of an ATP-dependent carboxylase in a CO2-dependent pathway of acetone metabolism by Xanthobacter strain Py2.

The metabolism of acetone by the aerobic bacterium Xanthobacter strain Py2 was investigated. Cell suspensions of Xanthobacter strain Py2 grown with propylene or glucose as carbon sources were unable to metabolize acetone. The addition of acetone to cultures grown with propylene or glucose resulted in a time-dependent increase in acetone-degrading activity. The degradation of acetone by these cultures was prevented by the addition of rifampin and chloramphenicol, demonstrating that new protein synthesis was required for the induction of acetone-degrading activity. In vivo and in vitro studies of acetone-grown Xanthobacter strain Py2 revealed a CO2-dependent pathway of acetone metabolism for this bacterium. The depletion of CO2 from cultures grown with acetone, but not glucose or n-propanol, prevented bacterial growth. The degradation of acetone by whole-cell suspensions of acetone-grown cells was stimulated by the addition of CO2 and was prevented by the depletion of CO2. The degradation of acetone by acetone-grown cell suspensions supported the fixation of 14CO2 into acid-stable products, while the degradation of glucose or beta-hydroxybutyrate did not. Cultures grown with acetone in a nitrogen-deficient medium supplemented with NaH13CO3 specifically incorporated 13C-label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-beta-hydroxybutyrate. Cell extracts prepared from acetone-grown cells catalyzed the CO2- and ATP-dependent carboxylation of acetone to form acetoacetate as a stoichiometric product. ADP or AMP were incapable of supporting acetone carboxylation in cell extracts. The sustained carboxylation of acetone in cell extracts required the addition of an ATP-regenerating system consisting of phosphocreatine and creatine kinase, suggesting that the carboxylation of acetone is coupled to ATP hydrolysis. Together, these studies provide the first demonstration of a CO2-dependent pathway of acetone metabolism for a strictly aerobic bacterium and provide direct evidence for the involvement of an ATP-dependent carboxylase in bacterial acetone metabolism.

Determination of the gene sequence and the three-dimensional structure at 2.4 angstroms resolution of methanol dehydrogenase from Methylophilus W3A1.

The DNA sequences for the genes encoding the heavy and light subunits of methanol dehydrogenase from Methylophilus methylotrophus W3A1 have been determined. The deduced amino acid sequence has enabled the structure of the enzyme to be refined at 2.4 angstrom resolution against X-ray data collected on a Hamlin area detector. The structure was refined using the programs PROFFT and X-PLOR with several model building step interspersed. The final model contains two heavy chains (571 amino acids), two light chains (69 amino acids), two molecules of pyrroloquinoline quinone, two Ca2+ and 521 solvent molecules. Each half molecule contains four disulfide linkages and four cis peptides. One of the disulfides is formed from two adjacent cysteine residues linked by a trans peptide which creates a novel eight-membered ring. The heavy subunit is an 8-fold beta-propeller, each "blade" of which is a four-stranded antiparallel twisted beta-sheet. The light chain is an elongated subunit stretching across the surface of the heavy subunit, with residues 1 to 32 containing four beta-turns and residues 33 to 62 forming a helix; however, it neither interacts with the active site, nor the other HL dimer and its functional role is obscure. Around the 8-fold beta-propeller there is a repeating pattern of tryptophan residues located in the outer strand of seven of the eight beta-leaflets, each packed between adjacent leaflets. Each of these tryptophan residues is centered in the beta-strand and participates in the main chain hydrogen bonding of the sheet. Five of the seven tryptophan residues have closely similar interactions with the adjacent beta-leaflet including stacking of the tryptophan indole rings against a peptide plane and formation of a hydrogen bond from NE1 of the indole ring to a main-chain carbonyl. This repeating pattern is conserved over a number of MEDH sequences. The PQQ is located on the pseudo 8-fold rotation axis of the heavy subunit, in a funnel-shaped internal cavity, sandwiched between the indole ring of Trp237 and the two sulfur atoms of the Cys103-Cys104 vicinal disulfide. A hexacoordinate Ca2+ is bound in the active site by one nitrogen and five oxygen ligands, three from the PQQ and the others from two protein side-chains. In the active site an isolated solvent molecule is bound to the O5 of PQQ and to a nearby aspartate side-chain; its position may be the binding site for methanol. The aspartate might than serve as a general base for proton abstraction from the substrate hydroxyl. The C5 atom of PQQ could be activated by electrophilic catalysis by a nearby argenine side-chain or by the calcium ion bound to PQQ.