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PubMed2004

A catalytic antibody against a tocopherol cyclase inhibitor.

The cyclic ammonium cation 5 and its guanidinium analogue 4 are inhibitors of tocopherol cyclase. Monoclonal antibodies were raised against protein conjugates of the haptens 1-3 and screened for catalytic reactions with alkene 8, a short chain analogue of the natural substrate phytyl-hydroquinone 6, and its enol ether analogues 10a,b. Antibody 16E7 raised against hapten 3 was found to catalyze the hydrolysis of Z enol ether 10a to form hemiacetal 12 with an apparent rate acceleration of k(cat)/k(uncat)=1400. Antibody 16E7 also catalyzed the elimination of Kemp's benzisoxazole 59. The absence of cyclization in the reaction of enol ether 10a was attributed to the competition of water molecules for the oxocarbonium cation intermediate within the antibody binding pocket. Hapten and reaction design features contributing to this outcome are discussed. Antibody 16E7 provides the first example of a carboxyl group acting both as an acid in an intrinsically acid-catalyzed process and as a base in an intrinsically base-catalyzed process, as expected from first principles. In contrast to the many examples of general-acid-catalyzed processes known to be catalyzed by catalytic antibodies, the specific-acid-catalyzed cyclization of phytyl-hydroquinone 6 or its analogue 8 still eludes antibody catalysis.

Adjuvants, Immunologic↗
PubMed2004

Homocoenzyme B12 and bishomocoenzyme B12: covalent structural mimics for homolyzed, enzyme-bound coenzyme B12.

Efficient electrochemical syntheses of "homocoenzyme B(12)" (2, Co(beta)-(5'-deoxy-5'-adenosyl-methyl)-cob(III)alamin) and "bishomocoenzyme B(12)" (3, Co(beta)-[2-(5'-deoxy-5'-adenosyl)-ethyl]-cob(III)alamin) are reported here. These syntheses have provided crystalline samples of 2 and 3 in 94 and 77 % yield, respectively. In addition, in-depth investigations of the structures of 2 and 3 in solution were carried out and a high-resolution crystal structure of 2 was obtained. The two homologues of coenzyme B(12) (2 and 3) are suggested to function as covalent structural mimics of the hypothetical enzyme-bound "activated" (that is, "stretched" or even homolytically cleaved) states of the B(12) cofactor. From crude molecular models, the crucial distances from the corrin-bound cobalt center to the C5' atom of the (homo)adenosine moieties in 2 and 3 were estimated to be about 3.0 and 4.4 A, respectively. These values are roughly the same as those found in the two "activated" forms of coenzyme B(12) in the crystal structure of glutamate mutase. Indeed, in the crystal structure of 2, the cobalt center was observed to be at a distance of 2.99 A from the C5' atom of the homoadenosine moiety and the latter was found to be present in the unusual syn conformation. In solution, the organometallic moieties of 2 and 3 were shown to be rather flexible and to be considerably more dynamic than the equivalent group in coenzyme B(12). The homoadenosine moiety of 2 was indicated to occur in both the syn and the anti conformations.

Bacterial Proteins↗
PubMed2006

A new methodology for the synthesis of fluorinated exo-glycals and their time-dependent inhibition of UDP-galactopyranose mutase.

Fluorinated carbohydrates constitute a very important class of mechanistic probes for glycosyl-processing enzymes. In this study, we describe the first synthesis of fluorinated and phosphonylated exo-glycals and their corresponding nucleotide sugars in the galactofuranose series. The synthetic protocol that we have developed is a Selectfluor-mediated fluorination/elimination sequence on phosphonylated exo-glycals, and it offers a new entry into fluorinated carbohydrate chemistry. The challenging E/Z stereochemical assignment of the resulting tetrasubstituted alkenes, which bear an alkoxy, an alkyl, a fluoro, and a phosphonyl group, has been achieved through NMR experiments. The corresponding (E)- and (Z)-nucleotide fluorosugars have been prepared and tested as inhibitors of UDP-galactopyranose mutase (UGM). UGM is a flavoenzyme that catalyzes the isomerization of uridine diphosphate(UDP)-galactopyranose into UDP-galactofuranose, a key step of the biosynthesis of important mycobacterial cell-wall glycoconjugates. The two diastereomeric molecules were found to display time-dependent inactivation of UGM, as expected from preliminary results using non-fluorinated exo-glycal nucleotides. The inhibitory properties of the two fluorinated molecules led us to suggest that the inactivation mechanism proceeds through two-electron processes, despite the presence of the flavin cofactor within the UGM catalytic site.

Fluorine↗
PubMed2005

Stabilisation of methylene radicals by cob(II)alamin in coenzyme B12 dependent mutases.

Coenzyme B12 initiates radical chemistry in two types of enzymatic reactions, the irreversible eliminases (e.g., diol dehydratases) and the reversible mutases (e.g., methylmalonyl-CoA mutase). Whereas eliminases that use radical generators other than coenzyme B12 are known, no alternative coenzyme B12 independent mutases have been detected for substrates in which a methyl group is reversibly converted to a methylene radical. We predict that such mutases do not exist. However, coenzyme B12 independent pathways have been detected that circumvent the need for glutamate, beta-lysine or methylmalonyl-CoA mutases by proceeding via different intermediates. In humans the methylcitrate cycle, which is ostensibly an alternative to the coenzyme B12 dependent methylmalonyl-CoA pathway for propionate oxidation, is not used because it would interfere with the Krebs cycle and thereby compromise the high-energy requirement of the nervous system. In the diol dehydratases the 5'-deoxyadenosyl radical generated by homolysis of the carbon-cobalt bond of coenzyme B12 moves about 10 A away from the cobalt atom in cob(II)alamin. The substrate and product radicals are generated at a similar distance from cob(II)alamin, which acts solely as spectator of the catalysis. In glutamate and methylmalonyl-CoA mutases the 5'-deoxyadenosyl radical remains within 3-4 A of the cobalt atom, with the substrate and product radicals approximately 3 A further away. It is suggested that cob(II)alamin acts as a conductor by stabilising both the 5'-deoxyadenosyl radical and the product-related methylene radicals.

Cobamides↗
PubMed2007

Inhibitory effect of umbelliferone aminoalkyl derivatives on oxidosqualene cyclases from S. cerevisiae, T. cruzi, P. carinii, H. sapiens, and A. thaliana: a structure-activity study.

Eighteen coumarin derivatives were tested as inhibitors of oxidosqualene cyclases (OSCs) from Saccharomyces cerevisiae, Trypanosoma cruzi, Pneumocystis carinii, Homo sapiens, and Arabidopsis thaliana, all expressed in an OSC-defective strain of S. cerevisiae.35 All the compounds have an aminoalkyl chain bound to an aromatic nucleus; unconventional synthetic procedures (microwave- and ultrasound-promoted reactions) were successfully used to prepare some of them. The most interesting structure-dependent difference in inhibitory activities was observed with an N-oxide group replacement of the tertiary amino group at the end of the side chain. An interesting species specificity also emerged: T. cruzi OSC was the least sensitive enzyme; P. carinii and A. thaliana OSCs were the most sensitive. The remarkable activities of three compounds on the T. cruzi enzyme and of five of them on the P. carinii enzyme suggest the present series as a promising compound family for the development of novel antiparasitic agents.

Amines↗
PubMed2003

Mechanistic insights into oxidosqualene cyclizations through homology modeling.

2,3-Oxidosqualene cyclases (OSC) are key enzymes in sterol biosynthesis. They catalyze the stereoselective cyclization and skeletal rearrangement of (3S)-2,3-oxidosqualene to lanosterol in mammals and fungi and to cycloartenol in algae and higher plants. Sequence information and proposed mechanism of 2,3-oxidosqualene cyclases are closely related to those of squalene-hopene cyclases (SHC), which represent functional analogs of OSCs in bacteria. SHCs catalyze the cationic cyclization cascade converting the linear triterpene squalene to fused ring compounds called hopanoids. High stereoselectivity and precision of the skeletal rearrangements has aroused the interest of researchers for nearly half a century, and valuable data on studying mechanistic details in the complex enzyme-catalyzed cyclization cascade has been collected. Today, interest in cyclases is still unbroken, because OSCs became targets for the development of antifungal and hypocholesterolemic drugs. However, due to the large size and membrane-bound nature of OSCs, three-dimensional structural information is still not available, thus preventing a complete understanding of the atomic details of the catalytic mechanism. In this work, we discuss results gained from homology modeling of human OSC based on structural information of SHC from Alicyclobacillus acidocaldarius and propose a structural model of human OSC. The model is in accordance with previously performed experimental studies with mechanism-based suicide inhibitors and mutagenesis experiments with altered activity and product specificity. Structural insight should strongly stimulate structure-based design of antifungal or cholesterol-lowering drugs.

Amino Acid Sequence↗
PubMed2005

Proteomics of Shewanella oneidensis MR-1 biofilm reveals differentially expressed proteins, including AggA and RibB.

Shewanella oneidensis MR-1 is a Gram-negative, facultative aerobic bacterium, able to respire a variety of electron acceptors. Due to its capability to reduce solid ferric iron, S. oneidensis plays an important role in microbially induced corrosion of metal surfaces. Since this requires cellular adhesion to the metal surface, biofilm growth is an essential feature of this process. The goal of this work was to compare the global protein expression patterns of sessile and planktonic grown S. oneidensis cells by two-dimensional (2-D) gel electrophoresis. Mass spectrometry was used as an identification tool of the differentially expressed proteins. An IPG strip of pH 3-10 as well as pH 4-7 was applied for iso-electrofocusing. Analysis of the 2-D patterns pointed out a total of 59 relevant spots. Among these proteins, we highlight the involvement of a protein annotated as an agglutination protein (AggA). AggA is a TolC-like protein which is presumably part of an ABC transporter. Another differentially expressed protein is RibB, an enzyme of the riboflavin biosynthesis pathway. Riboflavin is the precursor molecule of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and may be necessary for the altered respiratory properties of the biofilm cells versus planktonic cells. Some proteins that were identified indicate an anaerobic state of the biofilm. This anaerobic way of living affects the energy gaining pathways of the cell and is reflected by the presence of several proteins, including those of a heme-utilization system.

Bacterial Proteins↗
PubMed1997

Crystallization and preliminary X-ray crystallographic analysis of squalene-hopene cyclase from Alicyclobacillus acidocaldarius.

The membrane-associated protein squalene-hopene cyclase from Alicyclobacillus acidocaldarius was overexposed in Escherichia coli and purified by ion exchange and gel permeation chromatography. Crystals of three interrelated forms were grown by vapor diffusion under identical conditions. The crystals diffract to about 2.3 A resolution, but they are unstable in the X-ray beam. An interpretable heavy-atom derivative was obtained.

Bacillaceae↗
PubMed2001

The role of radicals in enzymatic processes.

Research on the mechanism of action of coenzyme B12, adenosylcobalamin, as a graduate student introduced the author to the field of organic free radicals in enzymology. Twenty years later, related work on S-adenosylmethionine (SAM) as a "poor man's coenzyme B12" was initiated in a detailed analysis of the mechanism of action of lysine 2,3-aminomutase (LAM). The interconversion of L-lysine and L-beta-lysine is catalyzed by LAM, which requires SAM, pyridoxal-5'-phosphate (PLP), and a [4Fe-4S] cluster as coenzymes. The mechanism of this reaction has been delineated as a radical isomerization, in which radical formation is initiated by the [4Fe-4S]-dependent cleavage of the SAM into methionine and the 5'-deoxyadenosyl radical. The mechanism of this process is discussed, together with the role of this radical in hydrogen abstraction from lysine to initiate the substrate radical isomerization. The chemistry underlying the functions of SAM, PLP, and [4Fe-4S] in the action of LAM is novel in all respects, except for the formation of a lysine-PLP aldimine at the active site. Of the four free radicals in the mechanism, three have been characterized by EPR spectroscopy. In the suicide inactivation of adenosylcobalamin-dependent dioldehydrase (DDH) by glycolaldehyde, the formation of cob(II)alamin and 5'-deoxyadenosine is accompanied by the conversion of glycolaldehyde to cis-ethanesemidione radical at the active site. The cis-ethanesemidione radical has been characterized by EPR spectroscopy. Its exceptional stability at the active site is the basis for the inactivation of DDH by glycolaldehyde.

Animals↗
PubMed2004

Candida famata (Debaryomyces hansenii) DNA sequences containing genes involved in riboflavin synthesis.

Previously cloned Candida famata (Debaryomyces hansenii) strain VKM Y-9 genomic DNA fragments containing genes RIB1 (codes for GTP cyclohydrolase II), RIB2 (encodes specific reductase), RIB5 (codes for dimethylribityllumazine synthase), RIB6 (encodes dihydroxybutanone phosphate synthase) and RIB7 (codes for riboflavin synthase) were sequenced. The derived amino acid sequences of C. famata RIB genes showed extensive homology to the corresponding sequences of riboflavin synthesis enzymes of other yeast species. The highest identity was observed to homologues of D. hansenii CBS767, as C. famata is the anamorph of this hemiascomycetous yeast. The D. hansenii CBS767 RIB3 gene encoding specific deaminase was cloned. This gene successfully complemented riboflavin auxotrophy of the rib3 mutant of flavinogenic yeast, Pichia guilliermondii. Putative iron-responsive elements (potential sites for binding of the transcription factors Fep1p or Aft1p and Aft2p) were found in the upstream regions of some C. famata and D. hansenii RIB genes. The sequences of C. famata RIB genes have been submitted to the EMBL data library under Accession Nos AJ810169-AJ810173.

Amino Acid Sequence↗
PubMed1994

Metal requirements of the enzymes catalyzing conversion of glutamate to delta-aminolevulinic acid in extracts of Chlorella vulgaris and Synechocystis sp. PCC 6803.

In the biosynthetic conversion of glutamate to the tetrapyrrole precursor, delta-aminolevulinic acid (ALA), glutamate is activated at C-1 by glutamyl-tRNA synthetase-catalyzed ligation to tRNAGlu. Glutamyl-tRNA reductase next catalyzes reduction of the activated glutamate to glutamate-1-semialdehyde (GSA), which is then converted to ALA by GSA aminotransferase. Glutamyl-tRNA synthetase is known to require a divalent metal (usually Mg2+) for activity, but it has not been established whether Mg2+ or another metal ion is also required for glutamyl-tRNA reductase or GSA aminotransferase, because these enzymes have previously been assayed in combined incubations containing all factors required for conversion of glutamate to ALA. We now report the metal requirements individually for each of the three enzyme reactions. Glutamyl-tRNA reductase activity in extracts from both Chlorella vulgaris and Synechocystis sp. PCC 6803 was stimulated by Mg2+ and inhibited by EDTA. EDTA-pretreated Chlorella glutamyl-tRNA reductase-containing fraction had very little activity in the absence of added Mg2+, but recovered full activity in incubations containing added Mg2+. The divalent metal requirement could be met by Mg2+, Mn2+, or Ca2+. Maximum activity was reached at approximately 15 mM concentration of each of these metals, and higher concentrations were inhibitory. Zn2+ was inhibitory at micromolar concentrations. Chlorella glutamyl-tRNA synthetase showed a metal requirement that could be met by Mg2+ or Mn2+, but not Ca2+. Maximum activity was reached at approximately 15 mM Mg2+ or Mn2+. Although the presence of 10 mM Ca2+ did not affect the Mg2+ concentration optimum, Ca2+ increased the effectiveness of low concentrations of Mg2+. In contrast to glutamyl-tRNA synthetase and glutamyl-tRNA reductase, Chlorella GSA aminotransferase did not show a metal requirement or inhibition by EDTA. However, EDTA decreased nonenzymatic transformation of GSA to ALA.

Aldehyde Oxidoreductases↗
PubMed1999

Novel inhibitors of glutamyl-tRNA(Glu) reductase identified through cell-based screening of the heme/chlorophyll biosynthetic pathway.

The metabolite 5-aminolevulinic acid (ALA) is an early committed intermediate in the biosynthetic pathway of heme and chlorophyll formation. In plants, 5-aminolevulinic acid is synthesized via a two-step pathway in which glutamyl-tRNA(Glu) is reduced by glutamyl-tRNA(Glu) reductase (GluTR) to glutamate 1-semialdehyde, followed by transformation to 5-aminolevulinic acid catalyzed by glutamate 1-semialdehyde aminotransferase. Using an Escherichia coli cell-based high-throughput assay to screen small molecule libraries, we identified several chemical classes that specifically inhibit heme/chlorophyll biosynthesis at this point by demonstrating that the observed cell growth inhibition is reversed by supplementing the medium with 5-aminolevulinic acid. These compounds were further tested in vitro for inhibition of the purified enzymes GluTR and glutamate 1-semialdehyde aminotransferase as confirmation of the specificity and site of action. Several promising compounds were identified from the high-throughput screen that inhibit GluTR with an I(0.5) of less than 10 microM. Our results demonstrate the efficacy of cell-based high-throughput screening for identifying inhibitors of 5-aminolevulinic acid biosynthesis, thus representing the first report of exogenous inhibitors of this enzyme.

Aldehyde Oxidoreductases↗
PubMed2000

Radical mechanisms in adenosylmethionine- and adenosylcobalamin-dependent enzymatic reactions.

A class of enzymatic reactions of S-adenosylmethionine (AdoMet) has recently been recognized, in which AdoMet plays a novel role by initiating free radical formation through the intermediate formation of 5'-deoxyadenosine-5'-yl, the 5'-deoxyadenosyl radical. The reactions are in this way related to adenosylcobalamin-dependent processes, which also depend on the formation of the 5'-deoxyadenosyl radical as an intermediate. The mechanisms by which the 5'-deoxyadenosyl radical is generated by the AdoMet- and adenosylcobalamin-dependent enzymes are very different. However, the functions of the 5'-deoxyadenosyl radical are similar in that in all cases it abstracts hydrogen from a substrate to form 5'-deoxyadenosine and a substrate-derived free radical. In this paper, the role of the 5'-deoxyadenosyl radical in the reaction of the adenosylcobalamin-dependent reactions will be compared with its role in the AdoMet-dependent reaction of lysine 2,3-aminomutase. The mechanism by which AdoMet is cleaved to the 5'-deoxyadenosyl radical at enzymatic sites will also be discussed.

Binding Sites↗
PubMed2001

Inhibition of lysine 2,3-aminomutase by the alternative substrate 4-thialysine and characterization of the 4-thialysyl radical intermediate.

Lysine 2,3-aminomutase catalyzes the interconversion of L-lysine and L-beta-lysine. 4-Thia-L-lysine (4-thialysine) is an alternative substrate for Lysine 2,3-aminomutase. The organic free radical that appears in the steady state of the reaction of 4-thialysine is structurally analogous to the first lysine-based radical in the chemical mechanism (Wu, W., Lieder, K. W., Reed, G. H., and Frey, P. A. (1995) Biochemistry 34, 10532-10537). 4-Thialysine is a much more potent inhibitor of the reaction of lysine than would be anticipated on the basis of the value of Km for its reaction as a substrate. 4-Thialysine is here shown to be a competitive reversible inhibitor with respect to L-lysine, displaying an inhibition constant of 0.12 +/- 0.01 mM. The value of Km for 4-thialysine is 1.4 +/- 0.1 mM, and the maximum velocity Vm = 0.19 +/-0.02 micromol min(-1) mg-1 at 37 degrees C and pH 8.0. The kinetic parameters for the reaction of lysine under the same conditions are: Km = 4.2 +/- 0.5 mM and Vm = 43 +/- 1 micromol min(-1) mg(-1). The discrepancy between Km and the apparent Ki for 4-thialysine arises from the fact that the maximal velocity for 4-thialysine is only 0.44% that for L-lysine. The electron paramagnetic resonance spectra of the organic radical generated at the active site from 4-thialysine and those generated from deuterium and 3-13C-labeled forms of 4-thialysine were analyzed by simulation. Based on the resulting hyperfine splitting constants, the conformation and distribution of the unpaired spin of the radical at the active site were evaluated.

Binding, Competitive↗
PubMed1996

Enzymatic synthesis of UDP-galactofuranose and an assay for UDP-galactopyranose mutase based on high-performance liquid chromatography.

A method to prepare UDP-galactofuranose (UDP-Galf) free of UDP-galactopyranose (UDP-Galp) is described. The UDP-Galf is synthesized enzymatically from UDP-Galp using the enzyme UDP-galactopyranose mutase. Treatment of UDP-Galp with the enzyme yields an equilibrium mixture of UDP-Galp and UDP-Galf in which UDP-Galf is approximately 7%. In spite of its low yield, the UDP-Galf is readily purified from starting UDP-Galp using a Dionex PA-100 ion exchange HPLC column. The purified UDP-Galf was characterized by chemical degradations, by electrospray mass spectrometry, and by several nuclear magnetic resonance techniques. In addition, an HPLC assay for the enzyme UDP-galactopyranose mutase is presented that requires 0.5 microgram of UDP-Galf per assay and can be used for both qualitative and quantitative measurements of the enzyme activity. These procedures should thus aid in the characterization of the enzymes involved in galactofuranosyl biosynthesis for the cell walls of Mycobacteria, for the lipophosphoglycan of Leishmania, and for other microorganisms where galactofuranosyl residues are found.

Bacterial Proteins↗