Active-Site Mobility Revealed by the Crystal Structure of Arylmalonate Decarboxylase from Bordetella bronchiseptica

Kuettner, E.B.; Keim, Antje; Kircher, Markus; Rosmus, Susann; Sträter, Norbert

doi:10.1016/j.jmb.2007.12.069

Cited by 17 publications

(23 citation statements)

References 30 publications

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“…A Hammett study12 of the reaction found a ρ value of +1.19, which is consistent with a negatively charged transition state 10. Recently, preliminary X-ray diffraction experiments and an X-ray crystal structure of AMDase were reported 13,14…”

Section: Enantioselective Protonation In Enzymatic Systemssupporting

confidence: 54%

Enantioselective protonation

2009

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Enantioselective protonation is a common process in biosynthetic sequences. The decarboxylase and esterase enzymes that effect this valuable transformation are able to control both the steric environment around the proton acceptor (typically an enolate) and the proton donor (typically a thiol). Recently, several chemical methods to achieve enantioselective protonation have been developed by exploiting various means of enantiocontrol in different mechanisms. These laboratory transformations have proven useful for the preparation of a number of valuable organic compounds.A fundamental method to generate a tertiary carbon stereocenter is to deliver a proton to a carbanion intermediate. However, enantioselective transfer of a proton presents unusual challenges, specifically, manipulating a very small atom and avoiding product racemization at a particularly labile stereocenter. As a result, the conditions for a successful enantioselective protonation protocol may be very specific to a certain substrate class. Tertiary carbon stereocenters are extremely common in valuable biologically active natural products, and thus the need for synthetically useful enantioselective methods to form these stereocenters is vital. 1In this review, we discuss several strategic approaches to enantioselective protonation. Emphasis has been placed on recently developed methods and their accompanying mechanisms in order to update the most recent prior reviews on this topic. 2,3,4,5,6,7,8 Each method relies on particular stereochemical control elements based on the mechanism of the protonation transformation. Appreciation of these controlling elements may lead to improved methods for preparing valuable chiral materials for a variety of synthetic applications. Important Factors in Achieving Enantioselective ProtonationSeveral of the most important practical features of enantioselective protonation were enumerated in Fehr's 1996 review. 2 Principal among these is the fact that enantioselective protonations are necessarily kinetic processes since under thermodynamic control racemate would be formed. Accordingly, it is often necessary to match the pK a of the proton donor and the product to prevent racemization before product isolation. It is unfortunate that the same anion stabilizing groups (e.g., ketones) that make protonations relatively easy to achieve also impart a degree of instability in the product. This has led some researchers to explore hydrogen atom transfer reactions in lieu of Brønsted acid-mediated protonations (see the subsection Enantioselective Hydrogen Atom Transfer below).Correspondence should be addressed to B.M.S. stoltz@caltech.edu. The authors declare no competing financial interests. NIH Public Access Author ManuscriptNat Chem. Author manuscript; available in PMC 2010 April 27. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptIn addition to the obvious challenges of product stability under the reaction conditions, the rapid rate of proton exchange in solution often leads to significant l...

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Section: Enantioselective Protonation In Enzymatic Systemssupporting

confidence: 54%

Enantioselective protonation

2009

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“…Directed evolution of (S)-selective AMDase variant toward 1a by SSM Crystallographic [11][12][13] and biochemical 6,7,12) evidences in addition to recent computational calculations 14) have suggested a reaction mechanism for stereoselective decarboxylation, in which destabilization of the pro-R carboxyl residue by the hydrophobic pocket limits the rate of the whole catalytic process (Fig. 2).…”

Section: Resultsmentioning

confidence: 97%

“…Recently, we and another group have succeeded in determining the 3-D structure of AMDase by X-ray crystallography. [11][12][13] The structures indicated that AMDase catalyzes stereoselective decarboxylation of the pro-R carboxyl residue of the substrate, which is surrounded by aliphatic amino acid residues in the hydrophobic pocket, while the pro-S carboxyl residue is stabilized by six hydrogen bonds in the so-called dioxyanion hole. Accordingly, Okrasa et al suggested that this hydrophobic pocket destabilizes the carboxyl residue and initiates asymmetric decarboxylation (Fig.…”

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confidence: 99%

Engineered hydrophobic pocket of (S)-selective arylmalonate decarboxylase variant by simultaneous saturation mutagenesis to improve catalytic performance

Yoshida

Enoki

Kourist

et al. 2015

Bioscience, Biotechnology, and Biochemistry

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A bacterial arylmalonate decarboxylase (AMDase) catalyzes asymmetric decarboxylation of unnatural arylmalonates to produce optically pure (R)-arylcarboxylates without the addition of cofactors. Previously, we designed an AMDase variant G74C/C188S that displays totally inverted enantioselectivity. However, the variant showed a 20,000-fold reduction in activity compared with the wild-type AMDase. Further studies have demonstrated that iterative saturation mutagenesis targeting the active site residues in a hydrophobic pocket of G74C/C188S leads to considerable improvement in activity where all positive variants harbor only hydrophobic substitutions. In this study, simultaneous saturation mutagenesis with a restricted set of amino acids at each position was applied to further heighten the activity of the (S)-selective AMDase variant toward α-methyl-α-phenylmalonate. The best variant (V43I/G74C/A125P/V156L/M159L/C188G) showed 9,500-fold greater catalytic efficiency kcat/Km than that of G74C/C188S. Notably, a high level of decarboxylation of α-(4-isobutylphenyl)-α-methylmalonate by the sextuple variant produced optically pure (S)-ibuprofen, an analgesic compound which showed 2.5-fold greater activity than the (R)-selective wild-type AMDase.

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“…Whilst we were preparing this crystallization communication, the crystal structure of AMDase from Bordetella bronchiseptica was published (Kü ttner et al, 2008;PDB code 2vlb). The enzyme was crystallized under different conditions from those described above.…”

Section: Crystallization and X-ray Diffraction Experimentsmentioning

confidence: 99%

Crystallization and preliminary X-ray diffraction experiments of arylmalonate decarboxylase fromAlcaligenes bronchisepticus

et al. 2008

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Arylmalonate decarboxylase catalyses the enantioselective decarboxylation of -aryl--methylmalonates to produce optically pure -arylpropionates. The enzyme was crystallized with ammonium sulfate under alkaline pH conditions with the aim of understanding the mechanism of the enantioselective reaction. X-ray diffraction data collected to a resolution of 3.0 Å at cryogenic temperature showed that the crystals belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 83.13, b = 99.62, c = 139.64 Å . This suggested that the asymmetric unit would contain between four and six molecules. Small-angle X-ray scattering revealed that the enzyme exists as a monomer in solution. Thus, the assembly of molecules in the asymmetric unit was likely to have been induced during the crystallization process.

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Active-Site Mobility Revealed by the Crystal Structure of Arylmalonate Decarboxylase from Bordetella bronchiseptica

Cited by 17 publications

References 30 publications

Enantioselective protonation

Enantioselective protonation

Engineered hydrophobic pocket of (S)-selective arylmalonate decarboxylase variant by simultaneous saturation mutagenesis to improve catalytic performance

Crystallization and preliminary X-ray diffraction experiments of arylmalonate decarboxylase fromAlcaligenes bronchisepticus

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