Biomedical subjects
Publications and source records attributed to J B Spencer.
3,5-dihydroxyphenylacetate, a precursor for the non-proteinogenic amino acid 3,5-dihydroxyphenylglycine occurring in glycopeptide antibiotics, is determined to be catalysed by a type III polyketide synthase using malonyl-CoA as a starter unit.
The allylic hydrogen is found to have a dramatic effect on the regioselectivity of the Wacker oxidation, leading to the postulation that an agostic hydrogen or enyl (sigma + pi) complex helps to stabilise the key intermediate.
ORF22 from the chloroeremomycin gene cluster has been cloned, expressed and characterised as a hydroxymandelate oxidase (HmO) that is involved in the formation of both (S)-4-hydroxyphenylglycine and (S)-3,5-dihydroxyphenylglycine.
[figure: see text] A conceptually new palladium-catalyzed amidation reaction is described for the synthesis of beta-amido ketones based on derailing the Wacker oxidation of enones. This reaction generates a new carbon-nitrogen bond via a palladium-catalyzed conjugate addition of a carbamate nucleophile to an enone. The regiocontrol, mild and neutral conditions, lack of preactivation of the nucleophile, and lack of reoxidation system for the catalyst are attractive features of this transformation.
Pudgy (pu) homozygous mice exhibit clear patterning defects at the earliest stages of somitogenesis, resulting in adult mice with severe vertebral and rib deformities. By positional cloning and complementation, we have determined that the pu phenotype is caused by a mutation in the delta-like 3 gene (Dll3), which is homologous to the Notch-ligand Delta in Drosophila. Histological and molecular marker analyses show that the pu mutation disrupts the proper formation of morphological borders in early somite formation and of rostral-caudal compartment boundaries within somites. Viability analysis also indicates an important role in early development. The results point to a key role for a Notch-signalling pathway in the initiation of patterning of vertebrate paraxial mesoderm.
6-Methylsalicylic acid synthase, the multifunctional enzyme complex that catalyzes the biosynthesis of the tetraketide 6-methylsalicylic acid, was modified by thiol-specific inhibitors and cross-linking reagents. Treatment with 1,3-dibromopropan-2-one caused rapid enzyme inactivation and formation of cross-linked dimers. Analysis by SDS-PAGE, density gradient ultracentrifugation, and secondary modification with [14C]iodoacetamide showed that two types of cross-linked dimers were formed. Peptides derived from native and 1,3-dibromopropan[2-14C]one-treated enzyme were isolated by SDS-PAGE and N-terminally sequenced. The sequences of the two N-termini from cross-linked peptides were located in the nucleotide-derived amino acid sequence and found to arise from the beta-ketoacyl synthase and acyl carrier protein components of the 6-methylsalicylic acid synthase subunit. Acetyl-CoA protected the enzyme from both inactivation and cross-linking by binding to the reactive cysteine of the beta-ketoacyl synthase component. Malonyl-CoA protected against cross-linking by binding to the thiol moiety of the 4'-phosphopantetheine prosthetic group of the acyl carrier protein. Formation of a mixed disulfide on treatment with 5,5'-dithiobis(2-nitrobenzoic acid) indicated that these two types of thiol residue are positioned close to each other in the active enzyme. From these studies, it was concluded that two pairs of functional dimers are present in the 6-methylsalicylic acid synthase tetramer and that, within each dimer, the beta-ketoacyl synthase and acyl carrier protein components are juxtaposed to allow the respective cysteine (residue 204) and 4'-phosphopantetheine thiols to interact during condensation. This spatial arrangement of thiols at the condensing active site is analogous to that found in type I vertebrate fatty acid synthases and other polyketide synthases.
The first 12 enzymes involved in the biosynthesis of vitamin B12 from its five-carbon precursor, aminolevulinic acid, have been overexpressed in recombinant strains of Escherichia coli. The activity of each enzyme has been demonstrated by the biosynthesis of hydrogenobyrinic acid from aminolevulinic acid.
The C-terminus of the Escherichia coli CysG protein, consisting of amino acids 202-457, was expressed as a recombinant protein using gene dissection methodology. Analysis of the activity of this truncated protein, termed CysGA, revealed that it was able to methylate uroporphyrinogen III in the same S-adenosyl-L-methionine (SAM)-dependent manner as the complete CysG protein. However, this truncated protein was not able to complement E. coli cysG cells, thereby suggesting that the first 201 amino acids of the CysG protein had an enzymic activity associated with the conversion of dihydrosirohydrochlorin into sirohaem. Analysis of the N-terminus of the CysG protein revealed the presence of a putative pyridine dinucleotide binding site. When the purified CysG protein was incubated with NADP+, uroporphyrinogen III and SAM the enzyme was found to catalyse a coenzyme-mediated dehydrogenation to form sirohydrochlorin. The CysGA protein on the other hand showed no such coenzyme-dependent activity. Analysis of the porphyrinoid material isolated from strains harbouring plasmids containing the complete and truncated cysG genes suggested that the CysG protein was also involved in ferrochelation. The evidence presented in this paper suggests that the CysG protein is a multifunctional protein involved in SAM-dependent methylation, pyridine dinucleotide dependent dehydrogenation and ferrochelation.
BACKGROUND: Genetically engineered synthesis, in which the gene products, cofactors, and substrates of a complete pathway are combined in vitro in a single flask to give the target, can be a viable alternative to conventional chemical construction of molecules of complex structure and stereochemistry. We chose to attempt to synthesize the metal-free corrinoid hydrogenobyrinic acid, an advanced precursor of vitamin B12. RESULTS: Cloning and overexpression of the genes necessary for the S-adenosyl methionine dependent conversion of 5-aminolevulinic acid (ALA) to precorrin-3 and those required for the synthesis of hydrogenobyrinic acid from precorrin-3 completed the repertoire of the 12 biosynthetic enzymes involved in corrin synthesis. Using these enzymes and the necessary cofactors, the multi-enzyme synthesis of hydrogenobyrinic acid from ALA can be achieved in 20% overall yield in a single reaction vessel, corresponding to an average of at least 90% conversion for each of the 17 steps involved. CONCLUSIONS: By replacing the cell wall with glass, and by mixing the soluble biosynthetic enzymes and necessary cofactors, the major segment of the physiological synthesis of vitamin B12 has been accomplished. Since only those enzymes necessary for the synthesis of hydrogenobyrinic acid from ALA are supplied, none of the intermediates is deflected from the direct pathway. This results in an efficiency which in fact surpasses that of nature.
Previously, the E. coli cysG gene product had been shown to sequentially methylate uro'gen III to produce precorrin-2, hence it was given the trivial name uro'gen III methylase. We now report that in addition to methylase activity, the CysG protein catalyses both the NAD+ dependent oxidation of precorrin-2 to sirohydrochlorin, but also the insertion of iron into this oxidized intermediate, thereby producing siroheme. Thus CysG is a multifunctional protein solely responsible for siroheme synthesis from uro'gen III in E. coli, and accordingly is renamed siroheme synthase.
In the vitamin B12 biosynthetic pathway the enzymes responsible for the conversion of precorrin-3 to precorrin-4 have been identified as the gene products of cobG and cobJ from Pseudomonas denitrificans. CobG catalyzes the oxidation of precorrin-3 to precorrin-3x (a hydroxy lactone) whereas CobJ is a SAM-dependent C-17 methyl transferase and is necessary for ring contraction. A mechanism for ring contraction is proposed.
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6-Methylsalicylic acid synthase has been isolated in homogeneous form from Penicillium patulum grown in liquid culture from a spore inoculum. The enzyme is highly susceptible to proteolytic degradation in vivo and in vitro, but may be stabilized during purification by incorporating proteinase inhibitors in the buffers. The enzyme exists as a homotetramer of M(r) 750,000, with a subunit M(r) of 180,000. 6-Methylsalicyclic acid synthase also accepts acetoacetyl-CoA as an alternative starter molecule to acetyl-CoA. The enzyme also catalyses the formation of small amounts of triacetic acid lactone as an oligatory by-product of the reaction. In the absence of NADPH, triacetic acid lactone is the exclusive enzymic product, being formed at 10% of the rate of 6-methylsalicylic acid. The enzyme is inactivated by 1,3-dibromopropan-2-one, leading to the formation of cross-linked dimers similar to that observed with type I fatty acid synthases. Acetyl-CoA protects the enzyme against the inactivation and inhibits dimer formation. An adaptation of the purification method for 6-methylsalicylic acid synthase may be used for the isolation of fatty acid sythase from Penicillium patulum.
Chiral malonyl-CoA derivatives, enzymically synthesized from (R)- and (S)-[1-13C;2-2H]malonates using succinyl-CoA transferase, were incorporated into 6-methylsalicylic acid with homogeneous 6-methylsalicylic acid synthase isolated from Penicillium patulum. Analysis of the 6-methylsalicylic acid formed established that the hydrogen atoms at the 3- and 5-positions are derived from opposite absolute configurations in malonyl-CoA. When acetoacetyl-CoA was used as the starter molecule, a single hydrogen atom is incorporated from the chiral malonates into the 3-position of the 6-methylsalicylic acid. Mass spectrometric analysis of the 6-methylsalicylic acid indicates that this hydrogen atom originates from HRe of malonyl-CoA or HSi in the polyketide intermediate. It is thus concluded that the hydrogen atom at the 5-position of 6-methylsalicylic acid originates from HSi of malonyl-CoA or HRe in the polyketide intermediate. During the reaction the enzyme also catalyzes the stereospecific exchange of hydrogen atoms in the polyketide intermediates. The implications of the stereochemical information from these experiments are discussed in relation to the mechanism of the 6-methylsalicylic acid synthase reaction.
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Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.