Characterization of the Cereulide NRPS α-Hydroxy Acid Specifying Modules:  Activation of α-Keto Acids and Chiral Reduction on the Assembly Line

2006 ◽  
Vol 128 (33) ◽  
pp. 10698-10699 ◽  
Author(s):  
Nathan A. Magarvey ◽  
Monika Ehling-Schulz ◽  
Christopher T. Walsh
Keyword(s):  
Hydroxy Acid ◽  
Assembly Line ◽  
Keto Acids

Protein Assembly Line Components in Prodigiosin Biosynthesis:  Characterization of PigA,G,H,I,J

2006 ◽  
Vol 128 (39) ◽  
pp. 12600-12601 ◽  
Author(s):  
Sylvie Garneau-Tsodikova ◽  
Pieter C. Dorrestein ◽  
Neil L. Kelleher ◽  
Christopher T. Walsh
Keyword(s):  
Assembly Line ◽  
Protein Assembly

THE ALKALOIDS OF FUMARIACEOUS PLANTS: VIII. CORYDALIS AUREA, WILLD. AND THE CONSTITUTION OF BICUCINE

1933 ◽  
Vol 9 (5) ◽  
pp. 436-442 ◽  
Author(s):  
R. H. F. Manske
Keyword(s):  
Empirical Formula ◽  
Hydroxy Acid ◽  
Phenolic Hydroxyl ◽  
High Concentration ◽  
Chemical Examination ◽  
Low Concentration ◽  
Isolation And Characterization ◽  
Stems And Leaves ◽  
Constitutional Analysis

The chemical examination of the alkaloids of Corydalis aurea has shown an unusual complexity and of the total of more than ten alkaloids thus far isolated only six are now described. The record deals chiefly with the stems and leaves of the plant in which protopine was present in exceptionally low concentration (0.025%). Equally exceptional is its high concentration m the roots (1.6%). l-Tetrahydropalmatine constituted the largest fraction of the remaining alkaloids, and its present isolation is the first on record although the d-form was previously known. Two new and well-characterized alkaloids, which have been named capaurine and capauridine, respectively, are isomeric and are best represented by the empirical formula C21H27O5N. Both contain one phenolic hydroxyl and four methoxyl groups, and yield on methylation non-phenolic bases which do not appear to be identical. The presence of two bases, bicuculline and bicucine, which were first recorded in this series of papers, has again been demonstrated.The constitutional analysis of the new bases, as well as the isolation and characterization of the minor alkaloids, is in progress.In an appended note it is shown that bicuculline and bicucine are closely related and interconvertible. The latter is the free γ-hydroxy acid of which the former is the lactone.

scholarly journals Characterization of SyrC, an Aminoacyltransferase Shuttling Threonyl and Chlorothreonyl Residues in the Syringomycin Biosynthetic Assembly Line

2007 ◽  
Vol 14 (1) ◽  
pp. 31-40 ◽  
Author(s):  
Gitanjali M. Singh ◽  
Frédéric H. Vaillancourt ◽  
Jun Yin ◽  
Christopher T. Walsh
Keyword(s):  
Assembly Line

scholarly journals Identification and Characterization of Epoxide Carboxylase Activity in Cell Extracts of Nocardia corallina B276

1998 ◽  
Vol 180 (8) ◽  
pp. 2072-2078 ◽  
Author(s):  
Jeffrey R. Allen ◽  
Scott A. Ensign
Keyword(s):  
Enzyme System ◽  
Time Dependent ◽  
High Level Expression ◽  
Carboxylase Activity ◽  
Cell Extracts ◽  
Keto Acids ◽  
Nocardia Corallina ◽  
High Level ◽  
Identification And Characterization

ABSTRACT The metabolism of aliphatic epoxides (epoxyalkanes) by the alkene-utilizing actinomycete Nocardia corallina B276 was investigated. Suspensions of N. corallina cells grown with propylene as the carbon source readily degraded propylene and epoxypropane, while suspensions of glucose-grown cells did not. The addition of propylene and epoxypropane to glucose-grown cells resulted in a time-dependent increase in propylene- and epoxypropane-degrading activities that was prevented by the addition of rifampin and chloramphenicol. The expression of alkene- and epoxide-degrading activities was correlated with the high-level expression of several polypeptides not present in extracts of glucose-grown cells. Epoxypropane and epoxybutane degradation by propylene-grown cell suspensions of N. corallina was stimulated by the addition of CO2 and inhibited by the depletion of CO2. Cell extracts catalyzed the carboxylation of epoxypropane to form acetoacetate in a reaction that was dependent on the addition of CO2, NAD+, and a reductant (NADPH or dithiothreitol). In the absence of CO2, epoxypropane was isomerized by cell extracts to form acetone at a rate approximately 10-fold lower than the rate of epoxypropane carboxylation. Methylepoxypropane was found to be a time-dependent, irreversible inactivator of epoxyalkane-degrading activity. These properties demonstrate that epoxyalkane metabolism in N. corallinaoccurs by a carboxylation reaction forming β-keto acids as products and provide evidence for the involvement in this reaction of an epoxide carboxylase with properties and cofactor requirements similar to those of the four-component epoxide carboxylase enzyme system of the gram-negative bacterium Xanthobacter strain Py2 (J. R. Allen and S. A. Ensign, J. Biol. Chem. 272:32121–32128, 1997). The addition of epoxide carboxylase component I fromXanthobacter strain Py2 to methylepoxypropane-inactivatedN. corallina extracts restored epoxide carboxylase activity, and the addition of epoxide carboxylase component II fromXanthobacter Py2 to active N. corallinaextracts stimulated epoxide isomerase rates to the same levels observed with the purified Xanthobacter system. Antibodies raised against Xanthobacter strain Py2 epoxide carboxylase component I cross-reacted with a polypeptide in propylene-grownN. corallina extracts with the same molecular weight as component I but did not cross-react with glucose-grown extracts. Together, these results suggest a common pathway of epoxyalkane metabolism for phylogenetically distinct bacteria that involves CO2 fixation and the activity of a multicomponent epoxide carboxylase enzyme system.

scholarly journals Characterization of the 2-Hydroxy-acid Dehydrogenase McyI, Encoded within the Microcystin Biosynthesis Gene Cluster of Microcystis aeruginosa PCC7806

2006 ◽  
Vol 282 (7) ◽  
pp. 4681-4692 ◽  
Author(s):  
Leanne A. Pearson ◽  
Kevin D. Barrow ◽  
Brett A. Neilan
Keyword(s):  
Gene Cluster ◽  
Microcystis Aeruginosa ◽  
Hydroxy Acid ◽  
Biosynthesis Gene Cluster ◽  
Glutamate Mutase ◽  
Acid Dehydrogenase ◽  
Methyl Aspartate ◽  
Catalyzed Reactions

The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesized non-ribosomally by the thio-template function of a large modular enzyme complex encoded within the 55-kb microcystin synthetase gene (mcy) cluster. The mcy gene cluster also encodes several stand-alone enzymes, putatively involved in the tailoring and export of microcystin. This study describes the characterization of the 2-hydroxy-acid dehydrogenase McyI, putatively involved in the production of d-methyl aspartate at position 3 within the microcystin cyclic structure. A combination of bioinformatics, molecular, and biochemical techniques was used to elucidate the structure, function, regulation, and evolution of this unique enzyme. The recombinant McyI enzyme was overexpressed in Escherichia coli and enzymatically characterized. The hypothesized native activity of McyI, the interconversion of 3-methyl malate to 3-methyl oxalacetate, was demonstrated using an in vitro spectrophotometric assay. The enzyme was also able to reduce α-ketoglutarate to 2-hydroxyglutarate and to catalyze the interconversion of malate and oxalacetate. Although NADP(H) was the preferred cofactor of the McyI-catalyzed reactions, NAD(H) could also be utilized, although rates of catalysis were significantly lower. The combined results of this study suggest that hepatotoxic cyanobacteria such as M. aeruginosa PCC7806 are capable of producing methyl aspartate via a novel glutamate mutase-independent pathway, in which McyI plays a pivotal role.

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