Synthesis of 4,4-Disubstituted<scp>L</scp>-Glutamic Acids by Enzymatic Transamination

Hélaine, Virgil; Rossi, Joël; Gefflaut, Thierry; Alaux, Sébastien; Bolte, Jean

doi:10.1002/1615-4169(200108)343:6/7<692::aid-adsc692>3.0.co;2-6

Cited by 36 publications

(12 citation statements)

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“…Since the α‐keto acid precursor of 12 can be obtained by an aldol condensation of two molecules of pyruvic acid, we speculated that CghB might be involved in the formation of 12 . To test this hypothesis, we synthesized 12 chemically by dimerizing pyruvic acid under a basic condition in aqueous solution, then transaminated the product with a glutamic oxalacetic transaminase using cysteinesulfinic acid as an amino group donor to obtain 12 and its diastereomers . Then, those compounds were fed to the cghB knockout strain of CGKW14, an engineered C. globosum strain capable of site‐specific homologous recombination .…”

Section: Detailed Biosynthetic Pathways Of Natural Productssupporting

confidence: 68%

Elucidation of Biosynthetic Pathways of Natural Products

Kishimoto

Tsunematsu

Sato

et al. 2017

The Chemical Record

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During the last decade, we have revealed biosynthetic pathways responsible for the formation of important and chemically complex natural products isolated from various organisms through genetic manipulation. Detailed in vivo and in vitro characterizations enabled elucidation of unexpected mechanisms of secondary metabolite biosynthesis. This personal account focuses on our recent efforts in identifying the genes responsible for the biosynthesis of spirotryprostatin, aspoquinolone, Sch 210972, pyranonigrin, fumagillin and pseurotin. We exploit heterologous reconstitution of biosynthetic pathways of interest in our study. In particular, extensive involvement of oxidation reactions is discussed. Heterologous hosts employed here are Saccharomyces cerevisiae, Aspergillus nidulans and A. niger that can also be used to prepare biosynthetic intermediates and product analogs by engineering the biosynthetic pathways using the knowledge obtained by detailed characterizations of the enzymes. (998 char.).

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Section: Detailed Biosynthetic Pathways Of Natural Productssupporting

confidence: 68%

Elucidation of Biosynthetic Pathways of Natural Products

Kishimoto

Tsunematsu

Sato

et al. 2017

The Chemical Record

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“…Preparation of Enzyme Substrates-HMG and CHA were synthesized chemically using published methods with some modifications (22). Racemic KHG and HOPA were synthesized enzymatically using HpaI according to previous methods (23).…”

Section: Methodsmentioning

confidence: 99%

Structural and Kinetic Characterization of 4-Hydroxy-4-methyl-2-oxoglutarate/4-Carboxy-4-hydroxy-2-oxoadipate Aldolase, a Protocatechuate Degradation Enzyme Evolutionarily Convergent with the HpaI and DmpG Pyruvate Aldolases

Wang

Mazurkewich

Kimber

et al. 2010

Journal of Biological Chemistry

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4-Hydroxy-4-methyl-2-oxoglutarate/4-carboxy-4-hydroxy-2-oxoadipate (HMG/CHA) aldolase from Pseudomonas putida F1 catalyzes the last step of the bacterial protocatechuate 4,5-cleavage pathway. The preferred substrates of the enzyme are 2-keto-4-hydroxy acids with a 4-carboxylate substitution. The enzyme also exhibits oxaloacetate decarboxylation and pyruvate ␣-proton exchange activity. Sodium oxalate is a competitive inhibitor of the aldolase reaction. The pH dependence of k cat /K m and k cat for the enzyme is consistent with a single deprotonation with pK a values of 8.0 ؎ 0.1 and 7.0 ؎ 0.1 for free enzyme and enzyme substrate complex, respectively. The 1.8 Å x-ray structure shows a four-layered ␣-␤-␤-␣ sandwich structure with the active site at the interface of two adjacent subunits of a hexamer; this fold resembles the RNase E inhibitor, RraA, but is novel for an aldolase. The catalytic site contains a magnesium ion ligated by Asp-124 as well as three water molecules bound by Asp-102 and Glu-199. A pyruvate molecule binds the magnesium ion through both carboxylate and keto oxygen atoms, completing the octahedral geometry. The carbonyl oxygen also forms hydrogen bonds with the guanadinium group of Arg-123, which site-directed mutagenesis confirms is essential for catalysis. A mechanism for HMG/CHA aldolase is proposed on the basis of the structure, kinetics, and previously established features of other aldolase mechanisms.

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“…[17] Because the cgh gene cluster possessed an aldolase-coding cghB , we proposed CghB to catalyze the aldol condensation of two molecules of pyruvic acid to yield the intermediate 4 , which can then be stereoselectively transaminated to form 5 (Figure 1A). To test this hypothesis, we prepared 5 and its diastereomers chemoenzymatically [18] and used them in feeding experiments with an engineered C. globosum strain, CGKW14 [19] (Supporting Methods). After confirming the loss of production of 1 by ΔcghB /CGKW14 (Figure S14 i vs. iii), we fed 5 to this strain to observe the recovery of the production of 1 (Figure 2).…”

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confidence: 99%

Involvement of Lipocalin‐like CghA in Decalin‐Forming Stereoselective Intramolecular [4+2] Cycloaddition

et al. 2015

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Understanding enzymatic Diels—Alder (DA) reactions that can form complex natural product scaffold is of considerable interest. Sch 210972 1, a potential anti-HIV fungal natural product, contains a decalin core that is proposed to form via a DA reaction. We identified the gene cluster responsible for the biosynthesis of 1 and heterologously reconstituted the biosynthetic pathway in Aspergillus nidulans to characterize the enzymes involved. Most notably, deletion of cghA resulted in a loss of stereoselective decalin core formation, yielding both an endo 1 and a diastereomeric exo adducts of the proposed DA reaction. Complementation with cghA restored the sole formation of 1. Density functional theory computation of the proposed DA reaction provided a plausible explanation of the observed pattern of product formation. Based on our study, we propose that lipocalin-like CghA is responsible for the stereoselective intramolecular [4+2] cycloaddition that forms the decalin core of 1.

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Synthesis of 4,4-DisubstitutedL-Glutamic Acids by Enzymatic Transamination

Cited by 36 publications

References 0 publications

Elucidation of Biosynthetic Pathways of Natural Products

Elucidation of Biosynthetic Pathways of Natural Products

Structural and Kinetic Characterization of 4-Hydroxy-4-methyl-2-oxoglutarate/4-Carboxy-4-hydroxy-2-oxoadipate Aldolase, a Protocatechuate Degradation Enzyme Evolutionarily Convergent with the HpaI and DmpG Pyruvate Aldolases

Involvement of Lipocalin‐like CghA in Decalin‐Forming Stereoselective Intramolecular [4+2] Cycloaddition

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