Monitoring the acetohydroxy acid synthase reaction and related carboligations by circular dichroism spectroscopy

Vinogradov, Michael; Kaplun, Alexander; Vyazmensky, Maria; Engel, Stanislav; Golbik, Ralph; Tittmann, Kai; Uhlemann, Kathrin; Meshalkina, L. E.; Barak, Ze’ev; Hübner, Gerhard; Chipman, David M.

doi:10.1016/j.ab.2005.03.049

Cited by 26 publications

(30 citation statements)

References 28 publications

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“…The progress curves measured at 278 nm allowed determination of the steady state linear velocities of HOAS-catalyzed production of (R)-HOA (Fig. 1B) (17). At longer times, the negative CD band decreased, consistent with earlier observations of decarboxylation of (R)-HOA to achiral 2-hydroxylevulinate and similar to the decarboxylation of acetolactate to acetoin or of tartronate semialdehyde to glycolaldehyde (17).…”

Section: Detection and Characterization Of Hoasupporting

confidence: 72%

“…) are comparable with those of other carboligases that use 2-ketoacid substrates, such as glyoxylate carboligase and isozymes of acetohydroxyacid synthase from various bacteria (17,25,(27)(28)(29)(30). The rates on the unactivated enzyme are similar to those of M. tuberculosis acetohydroxyacid synthase, whereas the activated rates are comparable with those of E. coli enzymes.…”

supporting

confidence: 62%

“…Earlier, 1 H NMR and LC-MS analyses of this reaction assigned the product as 2-hydroxy-3-oxoadipate, which is decarboxylated non-enzymatically to hydroxylevulinate (7). By analogy with carboligase reactions of other ThDP-dependent enzymes (16,17), we assigned the observed band to the (R)-HOA enantiomer. The progress curves measured at 278 nm allowed determination of the steady state linear velocities of HOAS-catalyzed production of (R)-HOA (Fig.…”

Section: Detection and Characterization Of Hoamentioning

confidence: 99%

“…1B) (17). At longer times, the negative CD band decreased, consistent with earlier observations of decarboxylation of (R)-HOA to achiral 2-hydroxylevulinate and similar to the decarboxylation of acetolactate to acetoin or of tartronate semialdehyde to glycolaldehyde (17). Fitting this latter curve to a first-order equation yielded a halflife of 28.9 min for (R)-HOA with a first-order rate constant for decarboxylation of (4 Ϯ 0.05) ϫ 10 Ϫ4 s…”

Section: Detection and Characterization Of Hoamentioning

confidence: 99%

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Influence of Allosteric Regulators on Individual Steps in the Reaction Catalyzed by Mycobacterium tuberculosis 2-Hydroxy-3-oxoadipate Synthase

Balakrishnan

Jordan

Nathan

2013

Journal of Biological Chemistry

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Background: 2-Hydroxy-3-oxoadipate synthase is predicted to be essential for growth of Mycobacterium tuberculosis and is subject to allosteric regulation. Results: The role of regulators was characterized by kinetics and detection of thiamin diphosphate-bound covalent intermediate distribution. Conclusion:AcCoA activates steps leading to a predecarboxylation intermediate; GarA chiefly inhibits postdecarboxylation steps. Significance: This novel regulatory regime in thiamin diphosphate enzymes provides new leads to inhibition.

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Section: Detection and Characterization Of Hoasupporting

confidence: 72%

supporting

confidence: 62%

Section: Detection and Characterization Of Hoamentioning

confidence: 99%

Section: Detection and Characterization Of Hoamentioning

confidence: 99%

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Influence of Allosteric Regulators on Individual Steps in the Reaction Catalyzed by Mycobacterium tuberculosis 2-Hydroxy-3-oxoadipate Synthase

Balakrishnan

Jordan

Nathan

2013

Journal of Biological Chemistry

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“…Kinetic data were determined using circular dichroism, the use of which has previously been reported as a convenient method for studies on ALDC. 10,20,21 Attempts to use a Voges-Proskauer assay or a coupled assay with generation of NAD + were less successful (Supporting Information). The CD assays required the use of enantiomerically-enriched substrate (S)-1, therefore a new synthetic approach to this molecule was developed through the use of an enzyme-catalyzed kinetic resolution of (2S,3S)-7, followed by oxidation (Supporting Information).…”

mentioning

confidence: 99%

Structure and Mechanism of Acetolactate Decarboxylase

et al. 2013

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Acetolactate decarboxylase catalyzes the conversion of both enantiomers of acetolactate to the (R)-enantiomer of acetoin, via a mechanism that has been shown to involve a prior rearrangement of the non-natural (R)-enantiomer substrate to the natural (S)-enantiomer. In this paper, a series of crystal structures of ALDC complex with designed transition state mimics are reported. These structures, coupled with inhibition studies and site-directed mutagenesis provide an improved understanding of the molecular processes involved in the stereoselective decarboxylation/protonation events. A mechanism for the transformation of each enantiomer of acetolactate is proposed.Acetolactate decarboxylase [EC 4.1.1.5] (ALDC) catalyzes the decarboxylation of (S)--acetolactate 1 (the natural substrate) and (S)--acetohydroxybutyrate 2 to (R)-acetoin 3 and (R)-3-hydroxypentan-2-one 4 respectively (Scheme 1).1-11 As well as having significance in the brewing process, 5 ALDC has been used for the synthesis of enantiomerically-pure diols. 6 An interesting feature of this enzyme is that it also catalyses, at a lower rate, the decarboxylation of the corresponding (R)-enantiomer of 1 to give (R)-3, and also the conversion of (R)--acetohydroxybutyrate 2 to (R)-2-hydroxypentan-3-one 5 (Scheme 1). [7][8][9][10][11] Scheme 1. Decarboxylation reactions catalyzed by ALDC.Through a series of very careful 13 C-labelling and circular dichroism (CD) studies by Crout and coworkers [7][8][9][10][11] and Hill et al 4 (Supplementary Information), it was established that the high stereoselectivity of the reactions of (S)-1 and (S)-2 arises from decarboxylation to an intermediate enediol (or enolate) which is subsequently protonated on the face opposite that from which carbon dioxide was lost, and at the carbon atom to which it was attached, i.e. resulting in overall inversion (Scheme 1).9,10 A similar stereoselective protonation of an enolate within a chiral enzyme environment following a decarboxylation has been reported in malonate decarboxylases. 12The results observed for the (R)-enantiomers of ALDC substrates, in contrast, proceed via prior migration of the carboxylate group to the adjacent carbon atom via a conformation in which the C-O bonds are in a syn conformation. 7,8 From (R)-1, this results in the formation of a-acetolactate of (S)-configuration, i.e. the natural substrate, which subsequently undergoes decarboxylation to (R)-acetoin 3. In the case of (R)-2, this rearrangement gives the structural isomer (S)-2-hydroxy-2-methyl-3-oxopentanoate, (S)-6, which then undergoes decarboxylation to the observed (R)-2-hydroxypentan-3-one (R)-5.7 Hence ALDC appears to catalyze both the stereoselective decarboxylation/protonation and the rearrangement reaction of the non-natural substrate.Due to a lack of X-ray crystallographic information on ALDC prior to our studies, less was known about the precise mechanism by which the enzyme directs the various reaction steps described above. Although during our analyses we became aware of a PDB d...

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Enzymatic C‐C Bond Formation, Asymmetric

Kara¹,

Liese²

2010

Encyclopedia of Industrial Biotechnology

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The environmental awareness has grown during the past years focusing on efficient utilization of energy, elimination of waste and avoidance of toxic and/or hazardous reagents.Biocatalysis fulfills the requirement of “green chemistry”; since enzymes are biodegradable catalysts which can effectively catalyze the reactions under mild conditions(ambient temperature, no need for high pressure, no extreme pH, no toxic solvents and no heavy metal catalysts).C–C bond formation reactions are fundamental to all of organic chemistry, and enzymes show promise for use in this field. A variety of versatile building blocks in organic and pharmaceutical chemistry can be synthesized by biocatalysts. In comparison to available chemical routes enzymes also provide chemo‐, regio‐, and stereoselective catalysis. Different enzyme classes are applied in the field of C–C bond formation to synthesize complex, aldol reactions, and cyanohydrin formations. Few applications are also found in ketol‐ and aldol transfer reactions. Furthermore, promiscuous activity of lipases can be applied in C–C bond formation reactions.

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Monitoring the acetohydroxy acid synthase reaction and related carboligations by circular dichroism spectroscopy

Cited by 26 publications

References 28 publications

Influence of Allosteric Regulators on Individual Steps in the Reaction Catalyzed by Mycobacterium tuberculosis 2-Hydroxy-3-oxoadipate Synthase

Influence of Allosteric Regulators on Individual Steps in the Reaction Catalyzed by Mycobacterium tuberculosis 2-Hydroxy-3-oxoadipate Synthase

Structure and Mechanism of Acetolactate Decarboxylase

Enzymatic C‐C Bond Formation, Asymmetric

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