A stereochemical study on the metabolism of isobutyrate in Pseudomonas putida

Ammonium[2-³H,1-¹⁴C]isobutyrate was converted by Pseudomonas putida ATCC 21244 into S(+)-β-hydroxyisobutyric acid (β-HIBA) with loss of the α-tritium atom. The recovered isobutyrate had the same ${}^3\text{H}{}^{14}\text{C}$ as the starting material. Ammonium (2S)-[3-¹³C]isobutyrate was synthesized and converted by P. putida into β-HIBA. The ¹³C-nmr of the corresponding methyl ester benzoate showed ¹³C enrichment in the hydroxymethyl carbon atom. The results therefore indicate that isobutyrate metabolism in this organism proceeds via an unsaturated intermediate (probably methacrylyl-CoA) formed by dehydrogenation of the 2-pro-S-methyl group of the precursor (isobutyryl-CoA). Hydration of the intermediate proceeds with addition of a proton at C-2 from the same side as the hydrogen removed in the dehydrogenation.     

Flash-induced FTIR difference spectra of the water oxidizing complex in moderately hydrated photosystem II core films: effect of hydration extent on S-state transitions

Differently hydrated films of photosystem II (PSII) core complexes from Synechococcus elongatus were prepared in a humidity-controlled infrared cell. The relative humidity was changed by a simple method of placing a different ratio of glycerol/water solution in the sealed cell. The extent of hydration of the PSII film was lowered as the glycerol ratio increased. FTIR difference spectra of the water oxidizing complex upon the first to sixth flashes were measured at 10 degrees C using these hydrated PSII films. The FTIR spectra (1800-1200 cm(-1)) of the PSII films hydrated using 20% and 40% glycerol/water showed basically the same features as those of the core sample in solution [Noguchi, T., and Sugiura, M. (2001) Biochemistry 40, 1497-1502], and the prominent peaks exhibited clear period four oscillation patterns. These observations indicate that the S-state cycle properly functions in these hydrated samples. In the PSII films less hydrated, however, the efficiencies of S-state transitions decreased as the extent of hydration was lowered. This tendency was more significant in the S2 --> S3 and S3 --> S0 transitions than in the S1 --> S2 and S0 --> S1 transitions, indicating that the reactions or movements of water molecules are more strongly coupled with the former two transitions than the latter two. The implication of this observation was discussed in light of the water oxidizing mechanism especially in respect to the steps of substrate incorporation and proton release. Furthermore, in the OH stretching region (3800-3000 cm(-1)) of the first-flash spectrum, a differential signal was observed at 3618/3585 cm(-1), which was previously found in the S2/S1 spectrum of a frozen sample at 250 K and assigned to the water vibrations [Noguchi, T., and Sugiura, M. (2000) Biochemistry 39, 10943-10949]. The fact that the signal appeared even in rather dehydrated PSII films at a physiological temperature (10 degrees C) supported the idea that this water is located in the close vicinity of the Mn cluster and directly involved in the water oxidizing reaction. The results also showed that moderate hydration of the PSII sample made the whole OH region measurable, escaping from absorption saturation by bulk water, and thus will be a useful technique to monitor the water reactions during the S-state cycle using FTIR spectroscopy.     

Iron-catalyzed reaction products of alpha-tocopherol with 1-palmitoyl-2-linoleoyl-3-sn-phosphatidylcholine (13S)-hydroperoxide

Alpha-tocopherol was reacted with 1-palmitoyl-2-[(9Z,11E)-(S)-13-hydroperoxy-9,11-octadecadienoyl]-3-sn-phosphatidylcholine (13-PLPC-OOH) in the presence of a lipid-soluble iron chelate, Fe(III) acetylacetonate, in methanol at 37 degrees C. The reaction product was isolated and identified as a mixture of 1-palmitoyl-2-[(10E)-(12S,13S)-9-(8a-dioxy-alpha-tocopherone)-12,13-epoxy-10-octadecenoyl]-3-sn-phosphatidylcholine and 1-palmitoyl-2-[(9Z)-(12S,13S)-11-(8a-dioxy-alpha-tocopherone)-12,13-epoxy-9-octadecenoyl]-3-sn-phosphatidylcholine (TOO-epoxyPLPC), in which the 12,13-epoxyperoxyl radicals derived from 13-PLPC-OOH attacked the 8a-position of the alpha-tocopheroxyl radical. The iron and ascorbate-catalyzed reaction of 13-PLPC-OOH with alpha-tocopherol in phosphatidylcholine (PC) liposomes was assessed by measuring the reaction products of alpha-tocopherol. When 13-PLPC-OOH and alpha-tocopherol were added in saturated dimyristoyl-PC liposomes, the products were TOO-epoxyPLPC, alpha-tocopherylquinone, and epoxy-alpha-tocopherylquinones. In 1-palmitoyl-2-linoleoyl-PC (PLPC) liposomes, alpha-tocopherol could react with both the 13-PLPC-OOH derived 12,13-epoxyperoxyl radicals and the PLPC-derived peroxyl radicals and formed the addition products together with alpha-tocopherylquinone and epoxy-alpha-tocopherylquinones. Therefore, the iron-catalyzed decomposition of phospholipid hydroperoxides primarily produces epoxyperoxyl radicals, which react with the 8a-carbon centered radical of alpha-tocopherol in liposomal systems.     

Cloning and sequencing of the coenzyme B(12)-binding domain of isobutyryl-CoA mutase from Streptomyces cinnamonensis, reconstitution of mutase activity, and characterization of the recombinant enzyme produced in Escherichia coli.

Isobutyryl-CoA mutase (ICM) catalyzes the reversible, coenzyme B(12)-dependent rearrangement of isobutyryl-CoA to n-butyryl-CoA, which is similar to, but distinct from, that catalyzed by methylmalonyl-CoA mutase. ICM has been detected so far in a variety of aerobic and anaerobic bacteria, where it appears to play a key role in valine and fatty acid catabolism. ICM from Streptomyces cinnamonensis is composed of a large subunit (IcmA) of 62.5 kDa and a small subunit (IcmB) of 14.3 kDa. icmB encodes a protein of 136 residues with high sequence similarity to the cobalamin-binding domains of methylmalonyl-CoA mutase, glutamate mutase, methyleneglutarate mutase, and cobalamin-dependent methionine synthase, including a conserved DXHXXG cobalamin-binding motif. Using IcmA and IcmB produced separately in Escherichia coli, we show that IcmB is necessary and sufficient with IcmA and coenzyme B(12) to afford the active ICM holoenzyme. The large subunit (IcmA) forms a tightly associated homodimer, whereas IcmB alone exists as a monomer. In the absence of coenzyme B(12), the association between IcmA and IcmB is weak. The ICM holoenzyme appears to comprise an alpha(2)beta(2)-heterotetramer with up to two molecules of bound coenzyme B(12). The equilibrium constant for the ICM reaction at 30 degrees C is 1.7 in favor of isobutyryl-CoA, and the pH optimum is near 7.4. The K(m) values for isobutyryl-CoA, n-butyryl-CoA, and coenzyme B(12) determined with an equimolar ratio of IcmA and IcmB are 57 +/- 13, 54 +/- 12, and 12 +/- 2 microM, respectively. A V(max) of 38 +/- 3 units/mg IcmA and a k(cat) of 39 +/- 3 s(-1) were determined under saturating molar ratios of IcmB to IcmA.     

Peroxisomal metabolism of propionic acid and isobutyric acid in plants

The subcellular sites of branched-chain amino acid metabolism in plants have been controversial, particularly with respect to valine catabolism. Potential enzymes for some steps in the valine catabolic pathway are clearly present in both mitochondria and peroxisomes, but the metabolic functions of these isoforms are not clear. The present study examined the possible function of these enzymes in metabolism of isobutyryl-CoA and propionyl-CoA, intermediates in the metabolism of valine and of odd-chain and branched-chain fatty acids. Using (13)C NMR, accumulation of beta-hydroxypropionate from [2-(13)C]propionate was observed in seedlings of Arabidopsis thaliana and a range of other plants, including both monocots and dicots. Examination of coding sequences and subcellular targeting elements indicated that the completed genome of A. thaliana likely codes for all the enzymes necessary to convert valine to propionyl-CoA in mitochondria. However, Arabidopsis mitochondria may lack some of the key enzymes for metabolism of propionyl-CoA. Known peroxisomal enzymes may convert propionyl-CoA to beta-hydroxypropionate by a modified beta-oxidation pathway. The chy1-3 mutation, creating a defect in a peroxisomal hydroxyacyl-CoA hydrolase, abolished the accumulation of beta-hydroxyisobutyrate from exogenous isobutyrate, but not the accumulation of beta-hydroxypropionate from exogenous propionate. The chy1-3 mutant also displayed a dramatically increased sensitivity to the toxic effects of excess propionate and isobutyrate but not of valine. (13)C NMR analysis of Arabidopsis seedlings exposed to [U-(13)C]valine did not show an accumulation of beta-hydroxypropionate. No evidence was observed for a modified beta-oxidation of valine. (13)C NMR analysis showed that valine was converted to leucine through the production of alpha-ketoisovalerate and isopropylmalate. These data suggest that peroxisomal enzymes for a modified beta-oxidation of isobutyryl-CoA and propionyl-CoA could function for metabolism of substrates other than valine.     

Novel coenzyme B12-dependent interconversion of isovaleryl-CoA and pivalyl-CoA

5'-Deoxyadenosylcobalamin (AdoCbl)-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently characterized a fusion protein that comprises the two subunits of the AdoCbl-dependent isobutyryl-CoA mutase flanking a G-protein chaperone and named it isobutyryl-CoA mutase fused (IcmF). IcmF catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA, whereas GTPase activity is associated with its G-protein domain. In this study, we report a novel activity associated with IcmF, i.e. the interconversion of isovaleryl-CoA and pivalyl-CoA. Kinetic characterization of IcmF yielded the following values: a K(m) for isovaleryl-CoA of 62 ± 8 μM and V(max) of 0.021 ± 0.004 μmol min(-1) mg(-1) at 37 °C. Biochemical experiments show that an IcmF in which the base specificity loop motif NKXD is modified to NKXE catalyzes the hydrolysis of both GTP and ATP. IcmF is susceptible to rapid inactivation during turnover, and GTP conferred modest protection during utilization of isovaleryl-CoA as substrate. Interestingly, there was no protection from inactivation when either isobutyryl-CoA or n-butyryl-CoA was used as substrate. Detailed kinetic analysis indicated that inactivation is associated with loss of the 5'-deoxyadenosine moiety from the active site, precluding reformation of AdoCbl at the end of the turnover cycle. Under aerobic conditions, oxidation of the cob(II)alamin radical in the inactive enzyme results in accumulation of aquacobalamin. Because pivalic acid found in sludge can be used as a carbon source by some bacteria and isovaleryl-CoA is an intermediate in leucine catabolism, our discovery of a new isomerase activity associated with IcmF expands its metabolic potential.     

Evidence for the accumulation of a stable intermediate in the nonenzymatic hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate.

Solutions of 5,10-methenyltetrahydropteroylglutamate can be converted to a stable hydrated adduct by heating solutions at 50 degrees C at pH values of 3-5 for several hours. The adduct is stable at pH values from 4 to 9 for hours, but at pH values below 2 it is converted to 5,10-methenyltetrahydropteroylglutamate and at pH values above 8 it is converted to 5-formyltetrahydropteroylglutamate. Arguments are presented that the adduct is (11R)-5,10-hydroxymethylenetetrahydropteroylglutamate formed from (11S)-5,10-hydroxymethylenetetrahydropteroylglutamate by formation of an ylide at C-11 which undergoes inversion of the electron pair to form the (11R) isomer. The (11R) hydrated adducted is believed to be the isomer of 5,10-methenyltetrahydropteroylglutamate referred to as anhydroleucovorin B by Cosulich et al. [Cosulich, D. C., Roth, B., Smith, J. M., Hultquist, M. E., & Parker, R. P. (1952) J. Am. Chem. Soc. 74, 3252-3263]. In addition, a new mechanism for the formation of 5-formyltetrahydropteroylglutamate from either 5,10-methenyltetrahydropteroylglutamate or 10-formyltetrahydropteroylglutamate via (11R)-5,10-hydroxymethylenetetrahydropteroylglutamate is proposed. A requirement for this pathway is that the formyl proton of 10-formyltetrahydropteroylglutamate exchange with solvent protons. The exchange of this formyl proton was observed at all pH values from 5.5 to 11.5 at a rate which exceeded by more than an order of magnitude the rate of formation of 5-formyltetrahydropteroylglutamate.     

Application of the reverse dept polarization-transfer pulse sequence to monitor in vitro and in vivo metabolism of 13C-ethanol by 1H-NMR spectroscopy

Using the reverse 13C----1H DEPT polarization-transfer pulse sequence the metabolism of 13C ethanol in vitro and in vivo has been monitored by 1H-NMR spectroscopy. Using yeast alcohol dehydrogenase, acetaldehyde, the hydrated form of acetaldehyde and acetate were identified as metabolites of [2-13C]-ethanol. The ratio of hydrated to free acetaldehyde was dependent upon the protein concentration of the reaction mixture. Binding of acetaldehyde in an irreversible Schiffs base resulted in optimal enzyme activity. Hepatocytes from rats fasted for 20 h, metabolised [1-13C] and [2-13C]ethanol in a linear fashion, but no [13C]acetaldehyde was detected. Metabolic integrity of the hepatocytes was confirmed with [2-13C]acetate. The addition of disulfiram (50 micron) to hepatocyte suspensions which had been incubated with [1-13C]ethanol, resulted in the resynthesis of [13C]ethanol. The amount of [13C]ethanol resynthesized under these conditions represents intracellular acetaldehyde whose concentration was in the range of 400-800 mumol/g wet weight of hepatocytes when 50 mM ethanol had been originally incubated with the hepatocyte suspension. These studies show how NMR-polarization transfer pulse sequences can be used to monitor the metabolism of 13C-ethanol in vivo, and provide a unique tool to measure in vivo concentrations of acetaldehyde. The studies also suggest that cytoplasmic aldehyde dehydrogenase may play a major role in hepatic ethanol metabolism.     

Novel B(12)-Dependent Acyl-CoA Mutases and Their Biotechnological Potential

The recent spate of discoveries of novel acyl-CoA mutases has engendered a growing appreciation for the diversity of 5'-deoxyadenosylcobalamin-dependent rearrangement reactions. The prototype of the reaction catalyzed by these enzymes is the 1,2 interchange of a hydrogen atom with a thioester group leading to a change in the degree of carbon skeleton branching. These enzymes are predicted to share common architectural elements: a Rossman fold and a triose phosphate isomerase (TIM)-barrel domain for binding cofactor and substrate, respectively. Within this family, methylmalonyl-CoA mutase (MCM) is the best studied and is the only member found in organisms ranging from bacteria to man. MCM interconverts (2R)-methylmalonyl-CoA and succinyl-CoA. The more recently discovered family members include isobutyryl-CoA mutase (ICM), which interconverts isobutyryl-CoA and n-butyryl-CoA; ethylmalonyl-CoA mutase, which interconverts (2R)-ethylmalonyl-CoA and (2S)-methylsuccinyl-CoA; and 2-hydroxyisobutyryl-CoA mutase, which interconverts 2-hydroxyisobutyryl-CoA and (3S)-hydroxybutyryl-CoA. A variant in which the two subunits of ICM are fused to a G-protein chaperone, IcmF, has been described recently. In addition to its ICM activity, IcmF also catalyzes the interconversion of isovaleryl-CoA and pivalyl-CoA. This review focuses on the involvement of acyl-CoA mutases in central carbon and secondary bacterial metabolism and on their biotechnological potential for applications ranging from bioremediation to stereospecific synthesis of C2-C5 carboxylic acids and alcohols, and for production of potential commodity and specialty chemicals.     

Quantitative observation of backbone disorder in native elastin

Elastin is a key protein in soft tissue function and pathology. Establishing a structural basis for understanding its reversible elasticity has proven to be difficult. Complementary to structure is the important aspect of flexibility and disorder in elastin. We have used solid-state NMR methods to examine polypeptide and hydrate ordering in both elastic (hydrated) and brittle (dry) elastin fibers and conclude (i) that tightly bound waters are absent in both dry and hydrated elastin and (ii) that the backbone in the hydrated protein is highly disordered with large amplitude motions. The hydrate was studied by (2)H and (17)O NMR, and the polypeptide by (13)C and (2)H NMR. Using a two-dimensional (13)C MAS method, an upper limit of S < 0.1 was determined for the backbone carbonyl group order parameter in hydrated elastin. For comparison, S approximately approximately 0.9 in most proteins. The former result is substantiated by two additional observations: the absence of the characteristic (2)H spectrum for stationary amides and "solution-like" (13)C magic angle spinning spectra at 75 degrees C, at which the material retains elasticity. Comparison of the observed shifts with accepted values for alpha-helices, beta-sheets, or random coils indicates a random coil structure at all carbons. These conclusions are discussed in the context of known thermodynamic properties of elastin and, more generally, protein folding. Because coacervation is an entropy-driven process, it is enhanced by the observed backbone disorder, which, we suggest, is the result of high proline content. This view is supported by recent studies of recombinant elastin polypeptides with systematic proline substitutions.     

On the mechanism of prostacyclin and thromboxane A2 biosynthesis

The present research describes studies which address the mechanism of prostacyclin (PGI2) and thromboxane A2 (TXA2) biosynthesis. In addition to prostaglandin H1 (PGH1), PGG2, PGH2, and PGH3, also 8-iso-PGH2, 13(S)-hydroxy-PGH2, and 15-keto-PGH2 were applied to determine the substrate specificities and kinetics of prostacyclin and thromboxane synthase in more detail. Human platelet thromboxane synthase converted PGH1, 8-iso-PGH2, 13(S)-hydroxy-PGH2 and 15-keto-PGH2 into the corresponding heptadecanoic acid (C17) plus malondialdehyde, whereas the thromboxane derivative was formed only from PGG2, PGH2, and PGH3 together with the corresponding C17 metabolite and malondialdehyde in a 1:1:1 ratio. In contrast, PGG2, PGH2, 13(S)-hydroxy-PGH2, 15-keto-PGH2 and PGH3 were almost completely isomerized to the corresponding prostacyclin derivative by bovine aortic prostacyclin synthase, whereas PGH1 and 8-iso-PGH2 only produced the corresponding C17 hydroxy acid plus malondialdehyde. Isotope-labeling experiments with [5,6,8,9,11,12,14,15-2H]PGH2 revealed complete retention of label and no isotope effect in the course of thromboxane biosynthesis, but the loss of one 2H atom at C-6 with an isotope effect of 1.20 during PGI2 formation. Prostacyclin and thromboxane synthase bind both 9,11-epoxymethano-PGF2 alpha and 11,9-epoxymethano-PGF2 alpha at the heme iron, but according to their difference spectra in opposite ways with respect to the 9- and 11-position. In agreement with published model studies, a cage radical mechanism is proposed for both enzymes according to which the initial radical process is terminated through oxidation of carbon-centered radicals by the iron-sulfur catalytic site, followed by ionic rearrangement to PGI2 or TXA2. Various Fe(III) model compounds as well as liver microsomes or cytochrome P-450CAM can also form small amounts of PGI2 and TXA2, but mainly yield 12(S)-hydroxy-5,8,10-heptadecatrienoic acid plus malondialdehyde probably by a radical fragmentation pathway.     

2H and 13C nuclear magnetic resonance study of N-palmitoylgalactosylsphingosine (cerebroside)/cholesterol bilayers.

13C- and 2H-NMR experiments were used to examine the phase behavior and dynamic structures of N-palmitoylgalactosylsphingosine (NPGS) (cerebroside) and cholesterol (CHOL) in binary mixtures. 13C spectra of 13C=O-labeled and 2H spectra of [7,7-2H2] chain-labeled NPGS as well as 3 alpha-2H1 CHOL indicate that cerebroside and CHOL are immiscible in binary mixtures at temperatures less than 40 degrees C. In contrast, at 40 degrees C < t < or = T(C) (NPGS), up to 50 mol% CHOL can be incorporated into melted cerebroside bilayers. In addition, 13C and 2H spectra of melted NPGS/CHOL bilayers show a temperature and cholesterol concentration dependence. An analysis of spectra obtained from the melted 13C=O NPGS bilayer phase suggests that the planar NH-C=O group assumes an orientation tilted 40 degrees-55 degrees down from the bilayer interface. The similarity between the orientation of the amide group relative to the bilayer interface in melted bilayers and in the crystal structure of cerebroside suggests that the overall crystallographic conformation of cerebroside is preserved to a large degree in hydrated bilayers. Variation of temperature from 73 degrees to 86 degrees C and CHOL concentration from 0 to 51 mol% results in small changes in this general orientation of the amide group. 2H spectra of chain-labeled NPGS and labeled CHOL in NPGS/CHOL bilayer demonstrate that molecular exchange between the gel and liquid-gel (LG) phases is slow on the 2H time scale, and this facilitates the simulation of the two component 2H spectra of [7,7-2H2]NPGS/CHOL mixtures. Simulation parameters are used to quantitate the fractions of gel and LG cerebroside. The quadrupole splitting of [7,7-2H2]NPGS/CHOL mixtures and 2H simulations allows the LG phase bilayer fraction to be characterized as an equimolar mixture of cerebroside and CHOL.     

Titration of the phase transition of phosphatidylserine bilayer membranes. Effects of pH, surface electrostatics, ion binding, and head-group hydration

The dependence of the gel-to-fluid phase transition temperature of dimyristoyl- and dipalmitoylphosphatidylserine bilayers on pH, NaCl concentration, and degree of hydration has been studied with differential scanning calorimetry and with spin-labels. On protonation of the carboxyl group (pK2app = 5.5), the transition temperature increases from 36 to 44 degrees C in the fully hydrated state of dimyristoylphosphatidylserine (from 54 to 62 degrees C for dipalmitoylphosphatidylserine), at ionic strength J = 0.1. In addition, at least two less hydrated states, differing progressively by 1 H2O/PS, are observed at low pH with transition temperatures of 48 and 52 degrees C for dimyristoyl- and 65 and 68.5 degrees C for dipalmitoylphosphatidylserine. On deprotonation of the amino group (pK3app = 11.55) the transition temperature decreases to approximately 15 degrees C for dimyristoyl- and 32 degrees C for dipalmitoylphosphatidylserine, and a pretransition is observed at approximately 6 degrees C (dimyristoylphosphatidylserine) and 21.5 degrees C (dipalmitoylphosphatidylserine), at J = 0.1. No titration of the transition is observed for the fully hydrated phosphate group down to pH less than or equal to 0.5, but it affinity for water binding decreases steeply at pH greater than or equal to 2.6. Increasing the NaCl concentration from 0.1 to 2.0 M increases the transition temperature of dimyristoyphosphatidylserine by approximately 8 degrees C at pH 7, by approximately 5 degrees at pH 13, and by approximately 0 degrees C at pH 1. These increases are attributed to the screening of the electrostatic titration-induced shifts in transition temperature. On a further increase of the NaCl concentration to 5.5 M, the transition temperature increases by an additional 9 degree C at pH 7, 13 degree C at pH 13, approximately 7 degree C in the fully hydrated state at pH 1, and approximately 4 and approximately 0 degree C in the two less hydrated states. These shifts are attributed to displacement of water of hydration by ion binding. From the salt dependence it is deduced that the transition temperature shift at the carboxyl titration can be accounted for completely by the surface charge and change in hydration of approximately 1 H2O/lipid, whereas that of the amino group titration arises mostly from other sources, probably hydrogen bonding. The shifts in pK (delta pK2 = 2.85, delta pK3 = 1.56) are consistent with a reduced polarity in the head-group region, corresponding to an effective dielectric constant epsilon approximately or equal to 30, together with surface potentials of psi congruent to -100 and -150 mV at the carboxyl and amino group pKs, respectively. The transition temperature of dimyristoylphosphatidylserine-water mixtures decreases by approximately 4 degree C each water/lipid molecule added, reaching a limiting value at a water content of approximately 9-10 H2O/lipid molecule.     

Purification and characterization of 2-methyl-branched chain acyl coenzyme A dehydrogenase, an enzyme involved in the isoleucine and valine metabolism, from rat liver mitochondria

2-Methyl-branched chain acyl-CoA dehydrogenase was purified to homogeneity from rat liver mitochondria. The native molecular weight of the enzyme was estimated to be 170,000 by gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis both with and without 2-mercaptoethanol, the enzyme showed a single protein band with Mr = 41,500, suggesting that this enzyme is composed of four subunits of equal size. Its isoelectric point was 5.50 +/- 0.2, and A1%280 nm was 12.5. This enzyme contained protein-bound FAD. The purified enzyme dehydrogenated S-2-methylbutyryl-CoA and isobutyryl-CoA with equal activity. The activities with each of these compounds were co-purified throughout the entire purification procedure. This enzyme also dehydrogenated R-2-methylbutyryl-CoA, but the specific activity was considerably lower (22%) than that for the S-enantiomer. The enzyme did not dehydrogenate other acyl-CoAs, including isovaleryl-CoA, propionyl-CoA, butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA, at any significant rate. Apparent Km and Vmax values for S-2-methylbutyryl-CoA were 20 microM and 2.2 mumol min-1 mg-1, respectively, while those for isobutyryl-CoA were 89 microM and 2.0 mumol min-1 mg-1 using phenazine methosulfate as an artificial electron acceptor. The enzyme was also active with electron transfer flavoprotein. Tiglyl-CoA and methacrylyl-CoA were identified as the reaction products from S-2-methylbutyryl-CoA and isobutyryl-CoA, respectively. 2-Ethylacrylyl-CoA was produced from R-2-methylbutyryl-CoA. Tiglyl-CoA competitively inhibited the activity with both S-2-methylbutyryl-CoA and isobutyryl-CoA with a similar Ki. The enzyme activity was also severely inhibited by several organic sulfhydryl reagents such as N-ethylmaleimide, p-hydroxymercuribenzoate, and methyl mercury iodide. The pattern and degree of inhibition were essentially identical for both substrates. The purified 2-methyl-branched chain acyl-CoA dehydrogenase was immunologically distinct from isovaleryl-CoA-, short chain acyl-CoA-, medium chain acyl-CoA-, or long chain acyl-CoA dehydrogenase.     

Solid-state (13)C NMR reveals effects of temperature and hydration on elastin.

Elastin is the principal protein component of the elastic fiber in vertebrate tissue. The waters of hydration in the elastic fiber are believed to play a critical role in the structure and function of this largely hydrophobic, amorphous protein. (13)C CPMAS NMR spectra are acquired for elastin samples with different hydration levels. The spectral intensities in the aliphatic region undergo significant changes as 70% of the water in hydrated elastin is removed. In addition, dramatic differences in the CPMAS spectra of hydrated, lyophilized, and partially dehydrated elastin samples over a relatively small temperature range (-20 degrees C to 37 degrees C) are observed. Results from other experiments, including (13)C T(1) and (1)H T(1 rho) measurements, direct polarization with magic-angle spinning, and static CP of the hydrated and lyophilized elastin preparations, also support the model that there is significant mobility in fully hydrated elastin. Our results support models in which water plays an integral role in the structure and proper function of elastin in vertebrate tissue.     

Biosynthesis of theobroxide and its related compounds, metabolites of Lasiodiplodia theobromae

Administration of (13)C labeled acetates ([1-(13)C], [2-(13)C] and [1,2-(13)C(2)] to Lasiodiplodia theobromae showed the tetraketide origins of both theobroxide, a potato-tuber inducing substance [1, (1S, 2R, 5S, 6R)-3-methyl-7-oxa-bicyclo[4.1.0]hept-3-en-2,5-diol]) and its carbonyldioxy derivative [2, (1S, 4R, 5S, 6R)-7,9-dioxa-3-methyl-8-oxobicyclo [4.3.0]-2-nonene-4,5-diol]. The incorporation of acetate-derived hydrogen into 1 and 2 was studied using [2-(2)H(3), 2-(13)C]acetate. Three and one deuterium atoms were incorporated at one methyl and epoxy carbons, respectively. The observed loss of deuterium atoms from the methyl group suggests a considerable amount of exchange from the methyl group of [2-(2)H(3), 2-(13)C]acetate during biosynthesis of 1 and 2. Incorporation of [1-(13)C]- and [1,2-(13)C(2)]acetates indicates the carbonyl carbon of the carbonyldioxy derivative is derived from the carboxy carbon of the precursor.     

The elimination of Salmonella typhimurium in sewage sludge by aerobic mesophilic stabilization and lime hydrated stabilization

This study observed the effects of two methods, aerobic mesophilic stabilization and lime hydrated stabilization of sewage sludge upon the survival of Salmonella typhimurium. Raw (primary) sludges from the mechanical biological municipal sewage treatment plant were used. Aerobic stabilization and lime hydrated stabilization were carried out in a laboratory fermentor. Aerobic stabilization was carried out in the mesophilic temperature range (from 25.70+/-0.40 to 37.82+/-1.38 degrees C). Lime hydrated was used at an amount of 10 kg/m(3) for the stabilization. Sludge samples were inoculated with a broth culture of S. typhimurium. Quantitative and qualitative examinations of the presence of S. typhimurium were carried out. Aerobic mesophilic stabilization caused elimination S. typhimurium within 48 h. The T(90) value of S. typhimurium was 6.66+/-0.20 h. During the lime hydrated stabilization pH values significantly increased from 5.66+/-0.07 to 12.12+/-0.02 (P<0.01). S. typhimurium was inactivated within 1h and the T(90) value was 0.19+/-0.01 h. Our study confirmed that the treatment of sewage sludge with lime hydrated was significantly more effective than the aerobic mesophilic stabilization, (P<0.01).     

On the mechanism of action of methylmalonyl-CoA mutase. Change of the steric course on isotope substitution

When (methyl-2H3)methylmalonyl-CoA was reacted with partially purified methylmalonyl-CoA mutase, 1H-NMR revealed that about 24% of the migrating deuterium was lost after 88% conversion. When [methyl-3H]methylmalonyl-CoA was incubated with highly purified methylmalonyl-CoA mutase, tritium exchange with the medium depended on added methylmalonyl-CoA epimerase. With highly purified preparations of methylmalonyl-CoA mutase, effective tritium exchange from [5'-3H]adenosylcobalamin to water required the addition of methylmalonyl-CoA epimerase and of substrate (e.g. succinyl-CoA). By addition of [14C]succinyl-CoA to a partially purified preparation of methylmalonyl-CoA mutase, it was shown that the mutase binds one substrate molecule very tightly. Coupling the mutase reaction with the transcarboxylase reaction and using variously labelled succinyl-CoA as substrate, revealed that only (2R)- and not (2S)-methylmalonyl-CoA will be formed by the mutase with a kinetic isotope effect of 3.5 using (2H4)succinyl-CoA. When (1-13C) propionyl-CoA was reacted with a mixture of highly purified methylmalonyl-CoA carboxylase, epimerase and mutase, 13C-NMR signals were obtained for the thioester carbonyl of succinyl-CoA (relative intensity 100%) and of methylmalonyl-CoA (5%) as well as for the carboxyl of free succinic acid (27%) and of succinyl-CoA (less than 4.5%). Thus very little, if any, migration of the CoA from one carboxyl to the other appears to take place. (1,4-13C2)Succinic acid and (1,4-13C2)succinyl-CoA were synthesised and their 13C-NMR chemical shifts were exactly determined. Evidence is provided for a strict stereospecificity of the mutase toward the (2R)-epimer of methylmalonyl-CoA and for an incomplete stereospecificity toward the two diastereotopic 3-H atoms of succinyl-CoA. The latter, combined with a high intramolecular isotope discrimination, causes rapid washing-out of the migrating 2H and 3H to water and slow washing-in from the medium. Whenever migration of protium from the sterically less preferred 3-pro(S)- position of succinyl-CoA occurs and simultaneously a heavy isotope is maneuvered from the migratable 3-pro(R)- position into the labile alpha-position of methylmalonyl-CoA, the substitution by the COSCoA group takes place with inversion of configuration. When the sterically preferred 3-pro(R)-hydrogen atom migrates, the previously reported stereochemical retention occurs. A mechanistic and stereochemical scheme is discussed that fully accounts for all observations.     

Transition-state stabilization by adenosine deaminase: 1,6-addition of water to purine ribonucleoside, the enzyme's affinity for 6-hydroxy-1,6-dihydropurine ribonucleoside, and the effective concentration of substrate water at the active site

Positions of equilibria of highly unfavorable addition reactions, whose products are present at concentrations below the limits of detection, can be determined from equilibria of combination of anionic nucleophiles with quaternized enamines. Applied to the newly prepared 1-methylpurinium ribonucleoside cation, this method yields approximate equilibrium constants of 2 X 10(-9) M-1 for addition of water and 4 X 10(-5) M-1 for addition of N-acetylcysteine to neutral purine ribonucleoside, in dilute aqueous solution. Positions of 13C magnetic resonances and UV absorption maxima of the above complexes and comparison with those of adenosine deaminase complexes strongly suggest that purine ribonucleoside is bound by adenosine deaminase as the 1,6 covalent hydrate, not as a covalently bonded complex formed by addition of a thiol group at the active site. The favorable position of equilibrium of the hydration reaction on the enzyme, together with its extremely unfavorable position in free solution, indicates that the effective activity of substrate water at the active site is in the neighborhood of 10(10) M. The Ki value of the active diastereomer of 6-hydroxy-1,6-dihydropurine ribonucleoside is estimated as 1.6 X 10(-13) M, more than 8 orders of magnitude lower than the apparent dissociation constants of enzyme complexes with the substrate adenosine or the product inosine. The enzyme's remarkable affinity for this hydrated species, which is vanishingly rare in free solution, seems understandable in terms of the hydrate's close resemblance to a hydrated intermediate approaching the transition state in direct water attack on adenosine.     

Structural changes of D1 C-terminal alpha-carboxylate during S-state cycling in photosynthetic oxygen evolution

Changes in the chemical structure of alpha-carboxylate of the D1 C-terminal Ala-344 during S-state cycling of photosynthetic oxygen-evolving complex were selectively measured using light-induced Fourier transform infrared (FTIR) difference spectroscopy in combination with specific [(13)C]alanine labeling and site-directed mutagenesis in photosystem II core particles from Synechocystis sp. PCC 6803. Several bands for carboxylate symmetric stretching modes in an S(2)/S(1) FTIR difference spectrum were affected by selective (13)C labeling of the alpha-carboxylate of Ala with l-[1-(13)C]alanine, whereas most of the isotopic effects failed to be induced in a site-directed mutant in which Ala-344 was replaced with Gly. Labeling of the alpha-methyl of Ala with l-[3-(13)C]alanine had much smaller effects on the spectrum to induce isotopic bands due to a symmetric CH(3) deformation coupled with the alpha-carboxylate. The isotopic bands for the alpha-carboxylate of Ala-344 showed characteristic changes during S-state cycling. The bands appeared prominently upon the S(1)-to-S(2) transition and to a lesser extent upon the S(2)-to-S(3) transition but reappeared at slightly upshifted frequencies with the opposite sign upon the S(3)-to-S(0) transition. No obvious isotopic band appeared upon the S(0)-to-S(1) transition. These results indicate that the alpha-carboxylate of C-terminal Ala-344 is structurally associated with a manganese ion that becomes oxidized upon the S(1)-to-S(2) transition and reduced reversely upon the S(3)-to-S(0) transition but is not associated with manganese ion(s) oxidized during the S(0)-to-S(1) (and S(2)-to-S(3)) transition(s). Consistently, l-[1-(13)C]alanine labeling also induced spectral changes in the low frequency (670-350 cm(-1)) S(2)/S(1) FTIR difference spectrum.