Biomedical subjects

V L Vilker

Publications and source records attributed to V L Vilker.

10 recordsLinked to original sources

PubMed2002

Chorismate lyase: kinetics and engineering for stability.

By removing the enolpyruvyl group from chorismate, chorismate lyase (CL) produces p-hydroxybenzoate (p-HB) for the ubiquinone biosynthetic pathway. We have analyzed CL by several spectroscopic and chemical techniques and measured its kinetic (kcat=1.7 s(-1), K(m)=29 microM) and product inhibition parameters (K(p)=2.1 microM for p-HB). Protein aggregation, a serious problem with wild type CL, proved to be primarily due to the presence of two surface-active cysteines, whose chemical modification or mutation (to serines) gave greatly improved solution behavior and minor effects on enzyme activity. CL is strongly inhibited by its product p-HB; for this reason activity and inhibition measurements were analyzed by both initial rate and progress curve methods. The results are consistent, but in this case where the stable enzyme-product complex rapidly becomes the predominant form of the enzyme, progress curve methods are more efficient. We also report inhibition measurements with several substrate and product analogs that give information on ligand binding interactions of the active site. The biological function of the unusual product retention remains uncertain, but may involve a mechanism of directed delivery to the membrane-bound enzyme that follows CL in the ubiquinone pathway.

Anthranilate Synthase↗

PubMed2001

The crystal structure of chorismate lyase shows a new fold and a tightly retained product.

The enzyme chorismate lyase (CL) catalyzes the removal of pyruvate from chorismate to produce 4-hydroxy benzoate (4HB) for the ubiquinone pathway. In Escherichia coli, CL is monomeric, with 164 residues. We have determined the structure of the CL product complex by crystallographic heavy-atom methods and report the structure at 1.4-A resolution for a fully active double Cys-to-Ser mutant and at 2.0-A resolution for the wild-type. The fold involves a 6-stranded antiparallel beta-sheet with no spanning helices and novel connectivity. The product is bound internally, adjacent to the sheet, with its polar groups coordinated by two main-chain amides and by the buried side-chains of Arg 76 and Glu 155. The 4HB is completely sequestered from solvent in a largely hydrophobic environment behind two helix-turn-helix loops. The extensive product binding that is observed is consistent with biochemical measurements of slow product release and 10-fold stronger binding of product than substrate. Substrate binding and kinetically rate-limiting product release apparently require the rearrangement of these active-site-covering loops. Implications for the biological function of the high product binding are considered in light of the unique cellular role of 4HB, which is produced by cytoplasmic CL but is used by the membrane-bound enzyme 4HB octaprenyltransferase.

Amino Acid Sequence↗

PubMed2000

Temperature dependence of the formal reduction potential of putidaredoxin.

Putidaredoxin (Pdx), a [2Fe-2S] redox protein of size M(r) 11,600, transfers two electrons in two separate steps from the flavin containing putidaredoxin reductase to the heme protein, cytochrome CYP101 in the P450cam catalytic cycle. It has recently come to light, through NMR measurements, that there can be appreciable differences in the Pdx conformational dynamics between its reduced and oxidized states. The redox reaction entropy, deltaS(0')rc = (S(0')Pdx(r)-S(0')Pdx(0)), as determined from measurements of the variation in formal potential with temperature, E0'(T), provides a measure of the strength of this influence on Pdx function. We designed a spectroelectrochemical cell using optically transparent tin oxide electrodes, without fixed or diffusible mediators, to measure E0'(T) over the temperature range 0-40 degrees C. The results indicate that the redox reaction entropy for Pdx is biphasic, decreasing from -213 +/- 27 J mol(-1) K(-1) over 0-27 degrees C, to -582 +/- 150 J mol(-1) K (-1) over 27-40 degrees C. These redox reaction entropy changes are significantly more negative than the changes reported for most cytochromes, although our measurement over the temperature interval 0-27 degrees C is in the range reported for other iron-sulfur proteins. This suggests that Pdx (and other ferredoxins) is a less rigid system than monohemes, and that redox-linked changes in conformation, and/or conformational dynamics, impart to these proteins the ability to interact with a number of redox partners.

Electrochemistry↗

PubMed1999

Comparison of backbone dynamics of oxidized and reduced putidaredoxin by 15N NMR relaxation measurements.

The backbone dynamics of uniformly 15N-labeled reduced and oxidized putidaredoxin (Pdx) have been studied by 2D 15N NMR relaxation measurements. 15N T1 and T2 values and 1H-15N NOEs have been measured for the diamagnetic region of the protein. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameters (S2), the effective correlation time for internal motions (tau e), and the 15N exchange broadening contributions (Rex) for each residue, as well as the overall correlation time (tau(m)). Order parameters for the reduced Pdx are generally higher than for the oxidized Pdx, and there is increased mobility on the microsecond to millisecond time scale for the oxidized Pdx, in comparison with the reduced Pdx. These results clearly indicate that the oxidized protein exhibits higher mobility than the reduced one, which is in agreement with the recently published redox-dependent dynamics studied by amide proton exchange. In addition, we observed very high T1/T2 ratios for residues 33 and 34, giving rise to a large Rex contribution. Residue 34 is believed to be involved in the binding of Pdx to cytochrome P450cam (CYP101). The differences in the backbone dynamics are discussed in relation to the oxidation states of Pdx, and their impact on electron transfer. The entropy change occurring on oxidation of reduced Pdx has been calculated from the order parameters of the two forms.

Amides↗

PubMed1998

Exploration of the structural environment of the iron-sulfur cluster in putidaredoxin by nitrogen-15 NMR spectroscopy of selectively labeled cysteine residues.

Putidaredoxin is a di-iron protein whose paramagnetic region is not well characterized by 1H detected NMR. We have studied the structure of this region in greater detail by directly observed 15N NMR of oxidized and reduced putidaredoxin preparations in which the six cysteine residues are selectively labeled with 15N. A new method for preparation of a stable form of reduced putidaredoxin has been developed for use in NMR. The 15N NMR spectra of the oxidized and reduced forms are characteristically different, and we have measured and compared 15N chemical shifts, spin-lattice relaxation times (T1), and chemical shift/temperature dependences for both forms. Evidence for localized valencies of the iron atoms in the reduced form is presented. From the 15N T1 values of the oxidized form, reduced distances of the cysteine backbone 15N nuclei from the center of the Fe2S2 cluster have been calculated. These distances are consistent with those calculated from X-ray crystal structure data for five ferredoxins, and confirm the structural similarity of the Fe2S2 clusters in putidaredoxin and in these ferredoxins in the oxidized state.

Amino Acid Sequence↗

PubMed1997

A direct electrode-driven P450 cycle for biocatalysis.

The large potential of redox enzymes to carry out formation of high value organic compounds motivates the search for innovative strategies to regenerate the cofactors needed by their biocatalytic cycles. Here, we describe a bioreactor where the reducing power to the cycle is supplied directly to purified cytochrome CYP101 (P450cam; EC 1.14.15.1) through its natural redox partner (putidaredoxin) using an antimony-doped tin oxide working electrode. Required oxygen was produced at a Pt counter electrode by water electrolysis. A continuous catalytic cycle was sustained for more than 5 h and 2,600 enzyme turnovers. The maximum product formation rate was 36 nmol of 5-exo-hydroxycamphor/nmol of CYP101 per min.

Bioreactors↗

PubMed1996

Tetralin as a substrate for camphor (cytochrome P450) 5-monooxygenase.

Camphor (cytochrome P450) 5-monooxygenase, originally isolated from the bacterium Pseudomonas putida PgG 786, catalyzes the essentially stereospecific conversion of tetralin (1,2,3,4-tetrahydronaphthalene) to (R)-1-tetralol ((R).(-)-1,2,3,4-tetrahydro-1-naphthol): tetralin(aq) + NADH(aq) + O2(aq) = (R)-1-tetralol(aq) + NAD(aq) + H2O(l). The ratio of the amount of (S)-1-tetralol to the amount of (R)-1-tetralol is small (approximately 0.04) and the reaction is essentially stereospecific. The reaction time-course plot indicates the formation of additional product(s) from the (R)-1-tetralol. It is found that the above reaction obeys Michaelis-Menten kinetics and that dimethyl sulfoxide, methanol, and p-dioxane serve as accelerators. Approximate values of a Michaelis constant Km, limiting rate Vmax, and catalytic constant kcat are obtained for this reaction under a specified set of conditions. It is shown by means of a thermochemical cycle calculation that the apparent equilibrium constant for this reaction is approximately 4 x 10(65) at T = 298.15 K and pH 7.3. Thus, this reaction is "irreversible" and, unless the enzyme system is inactivated, it will proceed in the direction of complete formation of 1-tetralol from tetralin. A detailed description of the preparation of the camphor (cytochrome P450) 5-monooxygenase enzyme system from recombinant microorganisms is given.

Camphor 5-Monooxygenase↗

PubMed1988

Analysis and application of substrate hydrolysis rates in indirect ELISA of a purified plant virus.

The transient colorimetric signal in a microtiter plate is used to quantify a purified plant virus, cowpea mosaic virus (CPMV), over five concentration decades in a single plate. The method involves the coating of the polystyrene microtiter plate wells directly with the CPMV antigen, followed by incubation with a rabbit-derived CPMV-specific antibody, and lastly by incubation with a commercially available antibody against rabbit immunoglobulin which has been pre-labeled with alkaline phosphatase. The rate of p-nitrophenylphosphate hydrolysis, both non-specific and that which was catalyzed by this enzyme, was measured spectrophotometrically at 405 nm. Enzyme-catalyzed hydrolysis rates followed first order kinetics at all antigen coating concentrations, and the 1 degree rate constants, which ranged from 2 X 10(-6) min-1 to 1 X 10(-3) min-1, were found to increase with increasing antigen concentration.

Antigens, Viral↗

PubMed1985

Characterization of bacteria by particle beam mass spectrometry.

A technique is described for detecting and characterizing bacteria on a single-particle basis by mass spectrometry. The method involves generation of a particle beam of single whole cells which are rapidly volatilized and ionized in vacuum in the ion source of a quadrupole mass spectrometer. The particle beam can be generated, with minimal sample handling, from a naturally occurring aerosol or from a solution of bacteria that can be dispersed as an aerosol. The mass spectrum is generated by successively measuring the average intensities of different mass peaks. The average intensity is obtained by measuring the ion intensity distribution at the particular mass (m/e) for ion pulses from more than 1,000 bacteria particles. Bacillus cereus, Bacillus subtilis, and Pseudomonas putida samples were analyzed to test the capability of the instrument for differentiating among species of bacteria. Significant ion-intensity information was produced over the m/e range of 50 to 300, an improvement over previous pyrolysis-mass spectrometry results. The complex mass spectra contained a few unique peaks which could be used for the differentiation of the bacteria. A statistical analysis of the variations in peak intensities among the three bacteria provided a quantitative measure of the reproducibility of the instrument and its ability to differentiate among bacteria. The technique could lead to a new rapid method for the analysis of microorganisms and could be used for the detection of airborne pathogens on a continuous, real-time basis.

Journal Article↗

PubMedDate not supplied

Improving the cytochrome P450 enzyme system for electrode-driven biocatalysis of styrene epoxidation.

Cytochrome P450 enzymes catalyze a vast array of oxidative and reductive biotransformations that are potentially useful for industrial and pharmaceutical syntheses. Factors such as cofactor utilization and slow reaction rates for nonnatural substrates limit their large-scale usefulness. This paper reports several improvements that make the cytochrome P450cam enzyme system more practical for the epoxidation of styrene. NADH coupling was increased from 14 to 54 mol %, and product turnover rate was increased from 8 to 70 min(-1) by introducing the Y96F mutation to P450cam. Styrene and styrene oxide mass balance determinations showed different product profiles at low and high styrene conversion levels. For styrene conversion less than about 25 mol %, the stoichiometry between styrene consumption and styrene oxide formation was 1:1. At high styrene conversion, a second doubly oxidized product, alpha-hydroxyacetophenone, was formed. This was also the exclusive product when Y96F P450cam acted on racemic, commercially available styrene oxide. The alpha-hydroxyacetophenone product was suppressed in reactions where styrene was present at saturating concentrations. Finally, styrene epoxidation was carried out in an electroenzymatic reactor. In this scheme, the costly NADH cofactor and one of the three proteins (putidaredoxin reductase) are eliminated from the Y96F P450cam enzyme system.

Camphor 5-Monooxygenase↗