Biomimetic self-condensation of malonates mediated by nucleosides.

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.

Decarboxylative Claisen condensation catalyzed by in vitro selected ribozymes.

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".

Structure/function studies on a S-adenosyl-L-methionine-dependent uroporphyrinogen III C methyltransferase (SUMT), a key regulatory enzyme of tetrapyrrole biosynthesis.

The crystallographic structure of the Pseudomonas denitrificans S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase (SUMT), which is encoded by the cobA gene, has been solved by molecular replacement to 2.7A resolution. SUMT is a branchpoint enzyme that plays a key role in the biosynthesis of modified tetrapyrroles by controlling flux to compounds such as vitamin B(12) and sirohaem, and catalysing the transformation of uroporphyrinogen III into precorrin-2. The overall topology of the enzyme is similar to that of the SUMT module of sirohaem synthase (CysG) and the cobalt-precorrin-4 methyltransferase CbiF and, as with the latter structures, SUMT has the product S-adenosyl-L-homocysteine bound in the crystal. The roles of a number of residues within the SUMT structure are discussed with respect to their conservation either across the broader family of cobalamin biosynthetic methyltransferases or within the sub-group of SUMT members. The D47N, L49A, F106A, T130A, Y183A and M184A variants of SUMT were generated by mutagenesis of the cobA gene, and tested for SAM binding and enzymatic activity. Of these variants, only D47N and L49A bound the co-substrate S-adenosyl-L-methionine. Consequently, all the mutants were severely restricted in their capacity to synthesise precorrin-2, although both the D47N and L49A variants produced significant quantities of precorrin-1, the monomethylated derivative of uroporphyrinogen III. The activity of these variants is interpreted with respect to the structure of the enzyme.

Mutagenesis identifies a conserved tyrosine residue important for the activity of uroporphyrinogen III synthase from Anacystis nidulans.

Uroporphyrinogen III synthase from the cyanobacterium Anacystis nidulans was overproduced in Escherichia coli and analyzed by site specific mutagenesis. Of the nine conserved amino acids altered, only a single tyrosine mutant (Y166F) showed any significant decrease in activity suggesting this residue is critical for proper substrate binding and/or catalysis.

Genetically engineered synthesis and structural characterization of cobalt-precorrin 5A and -5B, two new intermediates on the anaerobic pathway to vitamin B12: definition of the roles of the CbiF and CbiG enzymes.

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.

Genetically engineered production of 1-desmethylcobyrinic acid, 1-desmethylcobyrinic acid a,c-diamide, and cobyrinic acid a,c-diamide in Escherichia coli implies a role for CbiD in C-1 methylation in the anaerobic pathway to cobalamin.

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.

Identification and characterization of a novel vitamin B12 (cobalamin) biosynthetic enzyme (CobZ) from Rhodobacter capsulatus, containing flavin, heme, and Fe-S cofactors.

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.

Isolation and characterization of 14 additional genes specifying the anaerobic biosynthesis of cobalamin (vitamin B12) in Propionibacterium freudenreichii (P. shermanii).

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.

Structure of the Methanococcus jannaschii mevalonate kinase, a member of the GHMP kinase superfamily.

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.

Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium.

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.