Role of an Active Site Loop in the Promiscuous Activities of <i>Amycolatopsis</i> sp. T-1-60 NSAR/OSBS

McMillan, Andrew W.; Lopez, Mariana S.; Zhu, Mingzhao; Morse, Benjamin C.; Yeo, In-Cheol; Amos, Jaleesia; Hull, Kenneth G.; Romo, Daniel; Glasner, Margaret E.

doi:10.1021/bi500573v

Cited by 10 publications

(43 citation statements)

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“…However, it must be pointed out that mutations on the loop 20 of Amycolatopsis OSBS/NAAAR enzyme reduced both the synthase and the racemase activities, suggesting that evolution of this loop were not the only changes needed to evolve the latter activity. 57 As discussed below other residues contribute to accelerate the racemase activity in Amycolatopsis OSBS/NAAAR. We can analyze the contribution of the interaction energy of each residue to the barrier (calculated using Eq.…”

Section: Analysis Of Interactions Relevant For Catalysismentioning

confidence: 99%

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

et al. 2015

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The promiscuous activity of the enzyme o-Succinylbenzoate Synthase (OSBS) from the actinobacteria Amycolatopsis is investigated by means of QM/MM methods, using both Density Functional Theory and Semiempirical Hamiltonians. This enzyme catalyzes not only the dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate but also catalyzes racemization of different acylaminoacids, being N-succinyl-Rphenylglycine the best substrate. We investigated the molecular mechanisms for both reactions exploring the Potential Energy Surface. Then, Molecular Dynamics simulations were performed to obtain the free energy profiles and the averaged interaction energies of enzymatic residues with the reacting system. Our results confirm the plausibility of the reaction mechanisms proposed in the literature, with a good agreement between theoretical and experimentally-derived activation free energies. Our simulations unravel the role played by the different residues in each of the two possible reactions. The presence of flexible loops in the active site and the selection of structural modifications in the substrate seem to be key elements to promote the promiscuity of this enzyme.

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Section: Analysis Of Interactions Relevant For Catalysismentioning

confidence: 99%

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

et al. 2015

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“…After deciphering the natural function of NSAR, huge efforts have been paid on finding the molecular determinants allowing the evolution of the OSBS family and to understand the substrate promiscuity of NSAR/OSBS enzymes [69][70][71][72][73][74]. Protein…”

Section: Natural Function and Evolutionary Aspects On Nsar Enzymesmentioning

confidence: 99%

“…works have proved the difficulties to find the determinant allowing the evolution of OSBS family towards NSAR activity [70,71,74]. NSAR activity thus appears to be a recent evolutionary event towards new metabolic activities such as the interconversion of D-to L-amino acids for Geobacillus kaustophilus, which to date seems to be confined principally to enzymes in the Firmicutes NSAR/OSBS subfamily [57,58,74].…”

Section: Natural Function and Evolutionary Aspects On Nsar Enzymesmentioning

confidence: 99%

“…NSAR was already shown to be a member of the functional diverse enolase superfamily before the beginning of the new millennium [51]. Several works at the beginning of the 21 st century focused on deciphering the natural function of NSARs and some evolutionary aspects [51,57,58,[69][70][71][72][73][74][75][76] (see below), allowing to localize new NSARs in Bacillus/Geobacillus genera. Some of the NSARs belonging to this genera have been highly characterized and/or applied to the production of optically pure amino acids [27][28][29][30][31][32][33][34][35][36][37][38]59,[61][62][63][64][65][66][67][68].…”

Section: Introductionmentioning

confidence: 99%

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N-succinylamino acid racemases: Enzymatic properties and biotechnological applications

Martínez‐Rodríguez

Soriano-Maldonado

Gavira

2020

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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amino acid racemase; racemase; amino acid; enzymatic cascade Abbreviations: N-succinylamino acid racemase (NSAR); o-succinylbenzoate synthase (OSBS); N-succinylamino acid racemase/o-succinylbenzoate synthase subfamily (NSAR/OSBS); N-acetyl-amino or N-acyl-amino acid racemase (NAAR); Nsubstituted-amino acid (NxA); N-substituted-amino acid racemase (NxAR); Kinetic resolution (KR); Dynamic kinetic resolution (DKR); N-acetyl-(NA); N-acetyl-amino acid (NAA); N-succinyl-(NS); N-succinyl-amino acid (NSA); N-carbamoyl-(NC); Ncarbamoyl-amino acid (NCAA); N-chloroacetyl-(NCh); N-chloroacetyl-amino acid (NChAA); N-formyl-(NF); N-formyl-amino acid (NFAA); N-butyril (NBt); N-butyril amino acid (NBtA); N-propionyl (NPr); N-propionyl amino acid (NPrA); N-benzoyl (NBzA); N-benzoyl amino acid (NBzA); (1R,6R)-2-succinyl-6-hydroxy-2,4cyclohexadiene-1-carboxylate (SHCHC); Amycolatopsis sp. TS-1-60 NSAR (AmyNSAR); Geobacillus kaustophilus NSAR (GkaNSAR); Geobacillus stearothermophilus CECT 49 NSAR (GstNSAR); Streptomyces atratus Y-53 NSAR (SatNSAR); Sebekia benihana NSAR (SebeNSAR); Chloroflexus aurantiacus NSAR (CauNSAR); Thermus thermophilus NSAR (TteNSAR); Deinococcus radiodurans NSAR (DraNSAR); Exiguobacterium sp. AT1b OSBS (ExiOSBS); Alicyclobacillus acidocaldarius OSBS (AacOSBS); Enterococcus faecalis V583 NSAR (EfaNSAR); Listeria innocua Clip11262 NSAR (LinNSAR); Lysinibacillus varians GY32 NSAR (LvaNSAR); Roseiflexus castenholzii HLO8 NSAR (RcaNSAR); Amycolatopsis mediterranei S699 NSAR (AmedNSAR).

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“…[6][7][8] Studies of members of the enolase superfamily have afforded important insights into enzyme evolution and developing strategies for assigning the correct functions to proteins identified in genome projects. 3,[9][10][11][12] The well-characterized NOT THE PUBLISHED VERSION; this is the author's final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page.…”

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confidence: 99%

Inactivation of Mandelate Racemase by 3-Hydroxypyruvate Reveals a Potential Mechanistic Link between Enzyme Superfamilies

et al. 2015

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Mandelate racemase (MR), a member of the enolase superfamily, catalyzes the Mg 2+ -dependent interconversion of the enantiomers of mandelate. Several α-keto acids are modest competitive inhibitors of MR [e.g., mesoxalate (Ki = 1.8 ± 0.3 mM) and 3-fluoropyruvate (Ki = 1.3 ± 0.1 mM)], but, surprisingly, 3-hydroxypyruvate (3-HP) is an irreversible, time-dependent inhibitor (kinact/KI = 83 ± 8 M -1 s -1 ). Protection from inactivation by the competitive inhibitor benzohydroxamate, trypsinolysis and electrospray ionization tandem mass spectrometry analyses, and X-ray crystallographic studies reveal that 3-HP undergoes Schiff-base formation with Lys 166 at the active site, followed by formation of an aldehyde/enol(ate) adduct. Such a reaction is unprecedented in the enolase superfamily and may be a relic of an activity possessed by a promiscuous progenitor enzyme. The ability of MR to form and deprotonate a Schiff-base intermediate furnishes a previously unrecognized mechanistic link to other α/β-barrel enzymes utilizing Schiff-base chemistry and is in accord with the sequence-and structure-based hypothesis that members of the metal-dependent enolase superfamily and the Schiff-baseforming N-acetylneuraminate lyase superfamily and aldolases share a common ancestor.One of the first enzyme superfamilies recognized was the enolase superfamily, 1 whose members share a common partial reaction (i.e., the metal-assisted, Brønsted-base-catalyzed abstraction of the α-proton from a carboxylate substrate to form an enolate) 2-5 and a common protein fold [i.e., a modified (α/β)8-barrel (TIM barrel) bearing the catalytic residues at the C-terminal ends of the β-strands and an N-terminal domain that caps the active site conferring substrate specificity and excluding solvent]. [6][7][8] Studies of members of the enolase superfamily have afforded important insights into enzyme evolution and developing strategies for assigning the correct functions to proteins identified in genome projects. 3,[9][10][11][12] The well-characterized NOT THE PUBLISHED VERSION; this is the author's final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page.Biochemistry, Vol 54, No. 17 (May 5, 2015): pg. 2747-2757. DOI. This article is © American Chemical Society and permission has been granted for this version to appear in e-Publications@Marquette. American Chemical Society does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from American Chemical Society. 3archetype of this superfamily, mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida, catalyzes the Mg 2+ -dependent, 1,1-proton transfer reaction that interconverts the enantiomers of mandelate and has served as a useful paradigm for understanding how enzymes catalyze the rapid α-deprotonation of a carbon acid substrate with a relatively high pKa value. 13 MR utilizes a two-base mechanism with Lys 166 and His 297 abstracting the α-proton fr...

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Role of an Active Site Loop in the Promiscuous Activities of Amycolatopsis sp. T-1-60 NSAR/OSBS

Cited by 10 publications

References 64 publications

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

N-succinylamino acid racemases: Enzymatic properties and biotechnological applications

Inactivation of Mandelate Racemase by 3-Hydroxypyruvate Reveals a Potential Mechanistic Link between Enzyme Superfamilies

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