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1.
Biopolymers ; 88(1): 1-7, 2007.
Article in English | MEDLINE | ID: mdl-17054115

ABSTRACT

The insect kinins are present in a wide variety of insects and function as potent diuretic peptides, though they are subject to rapid degradation by internal peptidases. Insect kinin analogs incorporating stereochemical variants of (2S,4S)-4-aminopyroglutamate (APy), a cis-peptide bond motif, demonstrate significant activity in a cricket diuretic assay. Insect kinin analogs containing (2R,4R)-APy, (2S,4R)-APy and (2S,4S)-APy are essentially equipotent on an insect diuretic assay, with EC(50) values of about 10(-7)M, whereas the (2R,4S)-APy analog is at least 10-fold more potent (EC(50) = 7 x 10(-9)M). Conformational studies in aqueous solution indicate that the (2R,4S)-APy analog is considerably more flexible than the other three variants, which may explain its greater potency. The work identifies the optimal stereochemistry for the APy scaffold with which to design biostable, peptidomimetic analogs with the potential to disrupt critical insect kinin-regulated processes in insects.


Subject(s)
Insect Proteins/chemistry , Kinins/chemistry , Pyrrolidonecarboxylic Acid/analogs & derivatives , Amino Acid Motifs , Animals , Biopolymers/chemistry , Diuresis/drug effects , Drug Design , Female , Gryllidae/chemistry , Gryllidae/drug effects , Gryllidae/genetics , In Vitro Techniques , Insect Proteins/genetics , Insect Proteins/pharmacology , Insecta/chemistry , Insecta/drug effects , Insecta/genetics , Kinins/genetics , Kinins/pharmacology , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Pyrrolidonecarboxylic Acid/chemistry , Stereoisomerism
2.
Tetrahedron Lett ; 48(45): 8026-8028, 2007 Nov 05.
Article in English | MEDLINE | ID: mdl-18989357

ABSTRACT

Nucleoside mediated Claisen condensation of malonates has been achieved under biomimetic weak acid conditions, pH 3or 4, 0.15 M NaCl, and 0.125 M Mg(2+). The result illustrates the catalyzing property of end-nucleosides of t-RNA in the RNA world.

4.
J Am Chem Soc ; 128(30): 9971-8, 2006 Aug 02.
Article in English | MEDLINE | ID: mdl-16866557

ABSTRACT

Two new cobalt corrinoid intermediates, cobalt-precorrin 5A and cobalt-precorrin 5B, have been synthesized with the aid of overexpressed enzymes of the vitamin B(12) pathway of Salmonella entericaserovar typhimurium. These compounds were made in several regioselectively (13)C-labeled forms, and their structures have been established by multidimensional NMR spectroscopy. The addition of CbiF to the enzymes known to synthesize cobalt-precorrin 4 resulted in the formation of cobalt-precorrin 5A, and the inclusion of CbiG with CbiF produced cobalt-precorrin 5B, which has allowed us to define the role of these enzymes in the anaerobic biosynthetic pathway. CbiF is the C-11 methylase, and CbiG, an enzyme which shows homology with CobE of the aerobic pathway, is the gene product responsible for the opening of the ring A delta-lactone and extrusion of the "C(2)" unit. The discovery of these long-sought intermediates paves the way for defining the final stages of the anaerobic pathway. It is of considerable evolutionary interest that nature uses two distinct pathways to vitamin B(12), both conserved over several billion years and featuring completely different mechanisms for ring-contraction of the porphyrinoid to the corrinoid ring system. Thus the aerobic pathway utilizes molecular oxygen to trigger the events at C-20 leading to contraction and expulsion of the "C(2)" unit as acetic acid from a metal-free intermediate, whereas the anaerobic route features internal delivery of oxygen from a carboxylic acid terminus to C-20 followed by extrusion of the "C(2)" unit as acetaldehyde, using cobalt complexes as substrates.


Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Genetic Engineering , Organometallic Compounds/chemistry , Salmonella typhimurium/metabolism , Uroporphyrins/chemistry , Vitamin B 12/biosynthesis , Anaerobiosis , Models, Molecular , Molecular Structure , Vitamin B 12/chemistry
5.
Chem Commun (Camb) ; (13): 1439-41, 2006 Apr 07.
Article in English | MEDLINE | ID: mdl-16550293

ABSTRACT

The in vitro selection of RNAs catalyzing the decarboxylative Claisen condensation provides evidence for the synthesis of fatty acids, the building blocks of lipids and membranes, in the "RNA world".


Subject(s)
RNA, Catalytic/metabolism , Catalysis , Decarboxylation , Molecular Structure , RNA/chemistry , RNA/metabolism
6.
Bioorg Med Chem ; 14(11): 3904-22, 2006 Jun 01.
Article in English | MEDLINE | ID: mdl-16460949

ABSTRACT

The specificity toward substrate analogs of the first two methyltransferases in the vitamin B(12) biosynthetic pathway was probed with 15 synthetic porphyrinogens. Several novel methylated chlorins and isobacteriochlorins were isolated and characterized, suggesting the same methylation sequence C-2>C-7>C-20 as for the natural substrate, uro'gen III. The results allow us to narrow down possible structural requirements concerning substrate recognition by the methyltransferase enzymes.


Subject(s)
Methyltransferases/chemistry , Porphyrinogens/chemistry , Porphyrinogens/chemical synthesis , Vitamin B 12/biosynthesis , Magnetic Resonance Spectroscopy/methods , Magnetic Resonance Spectroscopy/standards , Methylation , Molecular Structure , Porphyrins/chemistry , Porphyrins/isolation & purification , Reference Standards , Sensitivity and Specificity , Stereoisomerism , Substrate Specificity
7.
Bioorg Med Chem ; 14(3): 724-31, 2006 Feb 01.
Article in English | MEDLINE | ID: mdl-16198574

ABSTRACT

Investigation on the use of the oxidized form (factor 3 (3a)) of the trimethylated intermediate (precorrin 3 (2)) as a substrate for the enzymes of the anaerobic pathway to vitamin B12 led to the synthesis of three pairs of novel cobalt corrinoids. The products were made with the aid of the Salmonella typhimurium enzymes CbiH, CbiF, CbiG, and CbiT, were synthesized in several 13C labeled versions, and were isolated as methylesters after esterification. Structures were determined by detailed NMR and MS analyses. Each set of products was obtained in the decarboxylated (RMe) and non-decarboxylated (R=CH2COOCH3) forms (at the C-12 position of the porphyrinoid).


Subject(s)
Corrinoids/biosynthesis , Corrinoids/chemistry , Vitamin B 12/biosynthesis , Anaerobiosis , Cobalt/chemistry , Magnetic Resonance Spectroscopy , Molecular Structure , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Salmonella typhimurium/enzymology , Salmonella typhimurium/genetics
8.
J Org Chem ; 70(24): 9997-10003, 2005 Nov 25.
Article in English | MEDLINE | ID: mdl-16292833

ABSTRACT

[reaction: see text] The cyclization of GGPP to taxadiene, catalyzed by taxadiene synthase, has been suggested to proceed through a series of monocyclic isocembrenyl- and bicyclic verticillyl-carbocationic intermediary stages. A set of GGPP analogues with abolished or perturbed pi-nucleophilicity at the delta10 double bond (GGPP numbering) was synthesized and incubated with taxadiene synthase to intercept the cyclization cascade at the monocyclic stage. Each analogue was transformed by taxadiene synthase in vitro to hydrocarbon products in varying yields, and the structures of the major product in each reaction were solved by GCEIMS and one- and two-dimensional (1H and 13C) NMR and found to be 14-membered monocyclic isocembrenyl diterpenes, indicating that the first C-C bond formation catalyzed by taxadiene synthase could be uncoupled from the other subsequent bond formation events by using suitably designed substrate analogues. The formation and isolation of these isocembrenyl diterpene products using taxadiene synthase supports proposals that the isocembrenyl cation is an intermediate in the cyclization of GGPP to taxadiene.


Subject(s)
Alkenes/chemical synthesis , Diterpenes/chemical synthesis , Isomerases/chemistry , Polyisoprenyl Phosphates/chemistry , Cyclization , Magnetic Resonance Spectroscopy/methods , Magnetic Resonance Spectroscopy/standards , Molecular Structure , Reference Standards , Stereoisomerism
9.
J Biol Chem ; 280(17): 16748-53, 2005 Apr 29.
Article in English | MEDLINE | ID: mdl-15741157

ABSTRACT

Co-expression of the cobA gene from Propionibacterium freudenreichii and the cbiA, -C, -D, -E, -T, -F, -G, -H, -J, -K, -L, and -P genes from Salmonella enterica serovar typhimurium in Escherichia coli resulted in the production of cobyrinic acid a,c-diamide. A cbiD deletion mutant of this strain produced 1-desmethylcobyrinic acid a,c-diamide, indicating that CbiD is involved in C-1 methylation in the anaerobic pathway to cobalamin. Strains that did not have the cbiP gene also produced 1-desmethylcobyrinic acid a,c-diamide, and strains that had neither cbiP nor cbiA synthesized 1-desmethylcobyrinic acid even in the presence of cbiD, suggesting that CbiA and CbiP are necessary for CbiD activity.


Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Genetic Engineering , Propionibacterium/metabolism , Vitamin B 12/analogs & derivatives , Vitamin B 12/chemistry , Vitamin B 12/genetics , Amides/chemistry , Cell Proliferation , Cobamides/chemistry , Escherichia coli/metabolism , Gene Deletion , Gene Expression Regulation, Bacterial , Methylation , Models, Chemical , Models, Molecular , Plasmids/metabolism , Porphyrinogens/chemistry , Salmonella typhimurium/metabolism , Spectrophotometry , Ultraviolet Rays , Vitamin B 12/biosynthesis , Vitamin B 12/metabolism
10.
J Biol Chem ; 280(2): 1086-94, 2005 Jan 14.
Article in English | MEDLINE | ID: mdl-15525640

ABSTRACT

One of the most intriguing steps during cobalamin (vitamin B12) biosynthesis is the ring contraction process that leads to the extrusion of one of the integral macrocyclic carbon atoms from the tetrapyrrole-derived framework. The aerobic cobalamin pathway requires the action of a monooxygenase called CobG (precorrin-3B synthase), which generates a hydroxylactone intermediate that is subsequently ring-contracted by CobJ. However, in the photosynthetic bacterium Rhodobacter capsulatus, which harbors an aerobic-like pathway, there is no cobG in the main cobalamin biosynthetic operon although it does contain an additional uncharacterized gene called orf663. To demonstrate the involvement of Orf663 in cobalamin synthesis, the first dedicated 10 genes of the B12 pathway (including orf663), encoding enzymes for the transformation of uroporphyrinogen III into hydrogenobyrinic acid (HBA), were sequentially cloned into a plasmid to generate an artificial operon, which, when transformed into Escherichia coli, endowed the host with the ability to make HBA. Deletion of orf663 from this operon prevented HBA synthesis, demonstrating that it was essential for corrin construction. HBA synthesis was restored to this recombinant strain either by returning orf663 or by substituting it with cobG. Recombinant overproduction of Orf663, now renamed CobZ, allowed the characterization of a novel cofactor-rich protein, housing two Fe-S centers, a flavin, and a heme group, which like B12 itself is a modified tetrapyrrole. A mechanism for Orf663 (CobZ) in cobalamin biosynthesis is proposed.


Subject(s)
Bacterial Proteins/metabolism , Coenzymes/metabolism , Flavins/metabolism , Heme/metabolism , Iron-Sulfur Proteins/metabolism , Rhodobacter capsulatus/enzymology , Vitamin B 12/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Coenzymes/chemistry , Electron Spin Resonance Spectroscopy , Escherichia coli/genetics , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/genetics , Molecular Structure , Oxidation-Reduction , Plasmids/genetics , Potentiometry , Rhodobacter capsulatus/genetics , Titrimetry
11.
Org Lett ; 5(24): 4713-5, 2003 Nov 27.
Article in English | MEDLINE | ID: mdl-14627422

ABSTRACT

[reaction: see text] Isoprenoid conjugates of nucleoside 5'-diphosphates were efficiently synthesized by one-step nucleophilic displacement reactions of either isoprenyl chlorides or isopentenyl tosylate with nucleoside 5'-diphosphates.


Subject(s)
Nucleotides/chemistry , Polyisoprenyl Phosphates/chemistry , Polyisoprenyl Phosphates/chemical synthesis , Magnetic Resonance Spectroscopy , Molecular Structure , Nucleotides/chemical synthesis
12.
J Org Chem ; 68(7): 2529-39, 2003 Apr 04.
Article in English | MEDLINE | ID: mdl-12662021

ABSTRACT

The chronology of the discoveries along the pathway of vitamin B(12) biosynthesis is reviewed from a personal perspective, including discussion of the most recent finding that two pathways to B(12) exist-one aerobic and one anaerobic-which differ mainly in the ring contraction mechanisms that convert porphyrin to corrin.


Subject(s)
Vitamin B 12 , Aerobiosis , Anaerobiosis , Bacteria/genetics , Bacteria/metabolism , Corrinoids , Cyclization , Magnetic Resonance Spectroscopy , Molecular Structure , Porphyrins/metabolism , Vitamin B 12/chemistry , Vitamin B 12/genetics , Vitamin B 12/metabolism
13.
Biochem J ; 370(Pt 2): 505-16, 2003 Mar 01.
Article in English | MEDLINE | ID: mdl-12408752

ABSTRACT

In Bacillus megaterium, the hemAXBCDL genes were isolated and were found to be highly similar to the genes from Bacillus subtilis that are required for the conversion of glutamyl-tRNA into uroporphyrinogen III. Overproduction and purification of HemC (porphobilinogen deaminase) and -D (uroporphyrinogen III synthase) allowed these enzymes to be used for the in vitro synthesis of uroporphyrinogen III from porphobilinogen. A second smaller cluster of three genes (termed sirABC) was also isolated and found to encode the enzymes that catalyse the transformation of uroporphyrinogen III into sirohaem on the basis of their ability to complement a defined Escherichia coli (cysG) mutant. The functions of SirC and -B were investigated by direct enzyme assay, where SirC was found to act as a precorrin-2 dehydrogenase, generating sirohydrochlorin, and SirB was found to act as a ferrochelatase responsible for the final step in sirohaem synthesis. CbiX, a protein found encoded within the main B. megaterium cobalamin biosynthetic operon, shares a high degree of similarity with SirB and acts as the cobaltochelatase associated with cobalamin biosynthesis by inserting cobalt into sirohydrochlorin. CbiX contains an unusual histidine-rich region in the C-terminal portion of the protein, which was not found to be essential in the chelation process. Sequence alignments suggest that SirB and CbiX share a similar active site to the cobaltochelatase, CbiK, from Salmonella enterica.


Subject(s)
Bacillus megaterium/enzymology , Heme/analogs & derivatives , Heme/biosynthesis , Uroporphyrins/metabolism , Vitamin B 12/biosynthesis , Amino Acid Sequence , Bacillus megaterium/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Methyltransferases/metabolism , Molecular Sequence Data , Salmonella typhimurium/enzymology , Salmonella typhimurium/genetics
14.
Microbiology (Reading) ; 148(Pt 6): 1845-1853, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12055304

ABSTRACT

A search for genes encoding enzymes involved in cobalamin (vitamin B12) production in the commercially important organism Propionibacterium freudenreichii (P. shermanii) has resulted in the isolation of an additional 14 genes encoding enzymes responsible for 17 steps of the anaerobic B12 pathway in this organism. All of the genes believed to be necessary for the biosynthesis of adenosylcobinamide from uroporphyrinogen III have now been isolated except two (cbiA and an as yet unidentified gene encoding cobalt reductase). Most of the genes are contained in two divergent operons, one of which, in turn, is closely linked to the operon encoding the B12-dependent enzyme methylmalonyl-CoA mutase. The close linkage of the three genes encoding the subunits of transcarboxylase to the hemYHBXRL gene cluster is reported. The functions of the P. freudenreichii B12 pathway genes are discussed, and a mechanism for the regulation of cobalamin and propionic acid production by oxygen in this organism is proposed.


Subject(s)
Genes, Bacterial/genetics , Propionibacterium/genetics , Propionibacterium/metabolism , Vitamin B 12/analogs & derivatives , Vitamin B 12/biosynthesis , Anaerobiosis , Base Sequence , Biological Transport , Cloning, Molecular , Cobalt/metabolism , Consensus Sequence , Methyltransferases/genetics , Molecular Sequence Data , Operon/genetics , Propionibacterium/enzymology , Uroporphyrinogens/metabolism , Vitamin B 12/metabolism
15.
J Biol Chem ; 277(11): 9462-7, 2002 Mar 15.
Article in English | MEDLINE | ID: mdl-11751891

ABSTRACT

The mevalonate-dependent pathway is used by many organisms to synthesize isopentenyl pyrophosphate, the building block for the biosynthesis of many biologically important compounds, including farnesyl pyrophosphate, dolichol, and many sterols. Mevalonate kinase (MVK) catalyzes a critical phosphoryl transfer step, producing mevalonate 5'-phosphate. The crystal structure of thermostable MVK from Methanococcus jannaschii has been determined at 2.4 A, revealing an overall fold similar to the homoserine kinase from M. jannaschii. In addition, the enzyme shows structural similarity with mevalonate 5-diphosphate decarboxylase and domain IV of elongation factor G. The active site of MVK is in the cleft between its N- and C-terminal domains. Several structural motifs conserved among species, including a phosphate-binding loop, have been found in this cavity. Asp(155), an invariant residue among MVK sequences, is located close to the putative phosphate-binding site and has been assumed to play the catalytic role. Analysis of the MVK model in the context of the other members of the GHMP kinase family offers the opportunity to understand both the mechanism of these enzymes and the structural details that may lead to the design of novel drugs.


Subject(s)
Methanococcus/enzymology , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Amino Acid Sequence , Binding Sites , Crystallization , Galactokinase/chemistry , Molecular Sequence Data , Phosphotransferases (Phosphate Group Acceptor)/chemistry , Protein Folding
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