On the steric course of the adenosylcobalamin-dependent 2-methyleneglutarate mutase reaction in Clostridium barkeri

The enzymatically active enantiomer of 3-methylitaconate in Clostridium barkeri has (R)-configuration. This was checked by fermentation of the racemate and reisolation of the (S)-enantiomer. In addition (R)-3-methylitaconate was synthesized by enzymatic isomerisation of 2,3-dimethylmaleate which was protonated at the Si-face. 2-Methylene[2-2H1]glutarate was synthesized via (R)-3-methyl[3-2H1]itaconate by brief incubation of 2,3-dimethylmaleate with a cell-free extract of Clostridium barkeri in 2H2O. The predominantly monodeuterated compound was oxidized to (S)-[2-2H1]succinate as analysed by circular dichroism. The results demonstrate that 2-methyleneglutarate mutase catalyses the reversible migration of an acryloyl residue from the alpha-carbon to the beta-carbon of propionate with inversion of configuration at the alpha-carbon.     

Interactions between bluefish and striped bass: Behavior of bluefish under size- and number-impaired conditions and overlap in resource use

A decline in bluefish (Pomatomus saltatrix L.) recreational landings during the 1990s and the early 2000s led to multiple theories on the ultimate cause. One theory was that a large portion of the bluefish population moved offshore and was unavailable to nearshore recreational fishers; one reason given for the movement offshore was increased competition with striped bass (Morone saxatilis W.). We conducted laboratory experiments (feeding and non-feeding) to examine behavioral interactions between adult bluefish and sub-adult striped bass in a large (121,000 L) research aquarium. Additionally, we examined diet and habitat overlap of bluefish and striped bass from the fall and spring bottom trawl surveys conducted by the National Marine Fisheries Service. Observations of feeding trials for the following treatments were made: non-impaired (i.e., same number and size of bluefish and striped bass), size-impaired (i.e., large striped bass/small bluefish), number-impaired (i.e.,10 striped bass/3bluefish), and single-species controls. Within a species, there was no difference in a variety of behavioral measures (e.g., attack rate, capture success, ingestion rate, and activity) between mixed- and control treatments under non-impaired or size-impaired conditions. However, behavior of number-impaired bluefish differed from control and size-impaired fish suggesting that striped bass may have a negative influence on bluefish foraging when bluefish are “out-numbered”. Feeding had a strong effect on swimming speeds for both species. Diet and habitat overlap between bluefish and striped bass in continental shelf waters was low. Overall, foraging behavior in mixed-species treatments and field observations suggest no competitive interactions between adult bluefish and sub-adult striped bass.     

Biochemical characterization of human 3-methylglutaconyl-CoA hydratase and its role in leucine metabolism

The metabolic disease 3-methylglutaconic aciduria type I (MGA1) is characterized by an abnormal organic acid profile in which there is excessive urinary excretion of 3-methylglutaconic acid, 3-methylglutaric acid and 3-hydroxyisovaleric acid. Affected individuals display variable clinical manifestations ranging from mildly delayed speech development to severe psychomotor retardation with neurological handicap. MGA1 is caused by reduced or absent 3-methylglutaconyl-coenzyme A (3-MG-CoA) hydratase activity within the leucine degradation pathway. The human AUH gene has been reported to encode for a bifunctional enzyme with both RNA-binding and enoyl-CoA-hydratase activity. In addition, it was shown that mutations in the AUH gene are linked to MGA1. Here we present kinetic data of the purified gene product of AUH using different CoA-substrates. The best substrates were (E)-3-MG-CoA (V(max) = 3.9 U.mg(-1), K(m) = 8.3 microM, k(cat) = 5.1 s(-1)) and (E)-glutaconyl-CoA (V(max) = 1.1 U.mg(-1), K(m) = 2.4 microM, k(cat) = 1.4 s(-1)) giving strong evidence that the AUH gene encodes for the major human 3-MG-CoA hydratase in leucine degradation. Based on these results, a new assay for AUH activity in fibroblast homogenates was developed. The only missense mutation found in MGA1 phenotypes, c.719C>T, leading to the amino acid exchange A240V, produces an enzyme with only 9% of the wild-type 3-MG-CoA hydratase activity.     

Purification of glutaryl-CoA dehydrogenase from Pseudomonas sp., an enzyme involved in the anaerobic degradation of benzoate

Cell-free extracts of Pseudomonas sp. strains KB 740 and K 172 both contained high levels of glutaryl-CoA dehydrogenase when grown anaerobically on benzoate or other aromatic compounds and with nitrate as electron acceptor. These aromatic compounds have in common benzoyl-CoA as the central aromatic intermediate of anerobic metabolism. The enzymatic activity was almost absent in cells grown aerobically on benzoate regardless whether nitrate was present. Glutaryl-CoA dehydrogenase activity was also detected in cell-free extracts of Rhodopseudomonas, Rhodomicrobium and Rhodocyclus after phototrophic growth on benzoate. Parallel to the induction of glutaryl-CoA dehydrogenase as measured with ferricenium ion as electron acceptor, an about equally high glutaconyl-CoA decarboxylase activity was detected in cell-free extracts. The latter activity was measured with the NAD-dependent assay, as described for the biotin-containing sodium ion pump glutaconyl-CoA decarboxylase from glutamate fermenting bacteria. Glutaryl-CoA dehydrogenase was purified to homogeneity from both Pseudomonas strains. The enzymes catalyse the decarboxylation of glutaconyl-CoA at about the same rate as the oxidative decarboxylation of glutaryl-CoA. The green enzymes are homotetramers (m=170 kDa) and contain 1 mol FAD per subunit. No inhibition was observed with avidin indicating the absence of biotin. The N-terminal sequences of the enzymes from both strains are similar (65%).     

Clostridium viride sp. nov., a strictly anaerobic bacterium using 5-aminovalerate as growth substrate,previously assigned to Clostridium aminovalericum

Strain T2–7, a 5-aminovalerate-fermenting bacterium previously classified as Clostridium aminovalericum, was further characterized, both physiologically and phylogenetically. Comparative sequencing analysis of the almost complete 16S rDNA revealed that strain T2–7 forms a distinct lineage within a phylogenetically coherent cluster of gram-positive bacteria currently assigned to the genus Clostridium. Strain T2–7 grew with 5-aminovalerate, 5-hydroxyvalerate, 4-hydroxybutyrate, vinylacetate, and crotonate, and required yeast extract and l-cysteine for growth. Other substrates were not utilized. The fermentation products, depending on the growth substrate, were ammonia, acetate, propionate, butyrate, and valerate. Sulphur was reduced by a mechanism not linked to energy conservation. Other acceptors were not utilized. Cells were gram-positive pointed-ended ovals, motile by means of two subpolar flagella, and possessed a gram-positive cell wall structure with an S-layer of hexagonally arranged subunits of 18.5 nm diameter. The DNA mol% G+C was 41.5. Strain T2–7 (DSM 6836) is proposed as the type strain of a new species, Clostridium viride sp. nov. Dedicated to H. A. Barker on the occasion of his 87th birthday     

The enzyme complex citramalate lyase from Clostridium tetanomorphum.

1. The enzyme citramalate from Clostridium tetanomorphum is not stable in crude extracts. However, the inactive enzyme can be reactivated by incubation with dithioerythritol followed by acetylation with acetic anhydride. Reactivation was also obtained with acetate, ATP, MgCl2 and acetate : SH-enzyme ligases (AMP) from C. tetanomorphum or Klebsiella aerogenes. 2. Incubation of the inactive enzyme with iodoacetate resulted in rapid loss of enzymic activity as determined by reactivation with acetic anhydride whereas the active enzyme was stable in the presence of iodoacetate. Using ido[2-(14)C]acetate the sites of carboxymethylation and acetylation where identified as cysteamine residues of the enzyme. The results demonstrate that the active enzyme contains acetyl thiolester residues which play the central role in the catalytic mechanism. 3. Citramalate lyase was purified by a procedure almost identical to that already described for citrate lyase from K. aerogenes. The molecular weight of citramalate lyase is equal to that of citrate lyase (Mr = 5.2--5.8 X 10(5)) as estimated by gel chromatography and sucrose gradient centrifugation. Polyacrylamide gel elctrophoresis of citramalate lyase in sodium dodecylsulfate yielded three polypeptide chains (Mr: alpha 5.3--5.6 X 10(4); beta 3.3--3.6 X 10(4); gamma 1.0--1.2 X 10(4)) in probably equal molar amounts. These data lead to a hexameric structure (alpha,beta,gamma)6 of the complete enzyme. 4. Pantothenate (5 mol/mol of enzyme) and the essential cysteamine residues were exclusively present in the gamma-chain, the acyl carrier protein of citramalate lyase. The acyl exchange and cleavage functions, probably catalysed by the alpha and beta-subunits, were measured with acyl-CoA derivatives which were able to substitute for the natural acyl carrier. 5. The results demonstrate that citramalate lyase is an enzyme complex with structure and functions closely resembling those of citrate lyase. Although the similarity between citramalate lyase and citrate lyases from various organisms suggests a close evolutionary relationship, these occur in very different, unrelated bacteria. A parallel situation found in the distribution of the nitrogenase system among procaryotes is discussed.     

Acetic anhydride: an intermediate analogue in the acyl-exchange reaction of citramalate lyase.

1. Reactivation of deacetyl citramalate lyase by acetic anhydride proceeds through an enzyme-anhydride complex prior to actual acetylation. The reaction is inhibited by citramalate which is competitive with acetic anhydride. 2. A corresponding complex is an intermediate in the carboxymethylation of deacetyl enzyme by iodoacetate. However, the inhibition of this reaction by S-citramalate appears to be non-competitive with iodoacetate. 3. The results lead to the conclusion that acetic anhydride can be regarded as a structural analogue of citramalic acetic anhydride, the proposed intermediate in the acyl exchange reaction on citramalate lyase. 4. The formation of 6-citryl thiolester from the 1-thiolester via the cyclic citric anhydride provides a chemicla model for enzymic acyl exchange. 5. The data suggest that anhydrides are of general importance in acyl exchange reactions of thiolesters.     

Identity of a gene responsible for suppression of aminoacyl-tRNA synthetase mutations with rpsT, the structural gene for ribosomal protein S20

Summary Specialized transducing lines of phage carrying segments between thr and car from the E. coli chromosome have been isolated. With help of these phages it has been shown that the gene sup _S20 (Böck et al., 1974) corresponds to rpsT, the structural gene for ribosomal protein S20.     

2-Hydroxyglutaryl-CoA dehydratase from Fusobacterium nucleatum (subsp. nucleatum): an iron-sulfur flavoprotein

Anaerobically prepared cell-free extracts from Fusobacterium nucleatum contain 2-hydroxyglutaryl-CoA dehydratase with a specific activity of 20 nkat mg^-1. The enzyme was purified 24-fold to a specific activity of 480 nkat mg^-1 by anion exchange chromatography, gel filtration and chromatography on Blue-Sepharose. The activity of the purified enzyme was strictly dependent on the reductant Ti(III)citrate and stimulated 25-fold by 0.15 mM ATP and 5 mM MgCl₂. ATP is hydrolysed to ADP during incubation with 2-hydroxyglutaryl-CoA dehydratase in the presence or absence of the substrate. The enzyme is extremely sensitive towards oxygen and is inhibited by 10 M chloramphenicol, 10 M 2,4-dinitrophenol or 0.15 mM hydroxylamine. The pure enzyme consists of three subunits (49 kDa), (39 kDa) and (24 kDa) in approximately equal amounts. In this respect the enzyme differs from the related 2-hydroxy-glutaryl-CoA dehydratase from Acidaminococcus fermentans and lactyl-CoA dehydratase from Clostridium propionicum both of which are composed of only two subunits with sizes comparable to those of and but require an additional protein for activity. The relative molecular mass of the native enzyme of about 100 kDa suggests a trimeric -structure. The homogeneous enzyme contains riboflavin (0.5 mol/112 kDa), iron and sulfur (3.5 mol/112 kDa each). Polyclonal antibodies directed against the 2-hydroxyglutaryl-CoA dehydratase from A. fermentans did not crossreact with cell free extracts or purified dehydratase from F. nucleatum. A comparison of the N-terminal amino acid sequences of the dehydratase subunits from A. fermentans and F. nucleatum, however, showed some similarities in the -subunits.Non-standard abbreviations DTT dithiothreitol - PAGE polyaccrylamide gel electrophoresis - VIS visible     

Oxygen exchange between acetate and the catalytic glutamate residue in glutaconate CoA-transferase from Acidaminococcus fermentans. Implications for the mechanism of CoA-ester hydrolysis.

The exchange of oxygen atoms between acetate, glutaryl-CoA, and the catalytic glutamate residue in glutaconate CoA-transferase from Acidaminococcus fermentans was analyzed using [(18)O(2)]acetate together with matrix-assisted laser desorption/ionization time of flight mass spectrometry of an appropriate undecapeptide. The exchange reaction was shown to be site-specific, reversible, and required both glutaryl-CoA and [(18)O(2)]acetate. The observed exchange is in agreement with the formation of a mixed anhydride intermediate between the enzyme and acetate. In contrast, with a mutant enzyme, which was converted to a thiol ester hydrolyase by replacement of the catalytic glutamate residue by aspartate, no (18)O uptake from H(2)(18)O into the carboxylate was detectable. This result is in accord with a mechanism in which the carboxylate of aspartate acts as a general base in activating a water molecule for hydrolysis of the thiol ester intermediate. This mechanism is further supported by the finding of a significant hydrolyase activity of the wild-type enzyme using acetyl-CoA as substrate, whereas glutaryl-CoA is not hydrolyzed. The small acetate molecule in the substrate binding pocket may activate a water molecule for hydrolysis of the nearby enzyme-CoA thiol ester.