Mutation of Aryl Binding Pocket Residues Results in an Unexpected Activity Switch in an <i>Oryza sativa</i> Tyrosine Aminomutase

Walter, Tyler; Wijewardena, Devinda P.; Walker, Kevin D.

doi:10.1021/acs.biochem.6b00331

Cited by 3 publications

(18 citation statements)

References 34 publications

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“…The enzyme also showed acceptance of phenylalanine, albeit with an activity just 3% of that for tyrosine, which possibly points to the evolutionary origin of this enzyme from an ancestral plant PAL. Building on this, variations identified at two positions in the active site of OsTAM1, and the related phenylalanine-specific TcPAM, allowed mutagenic studies of the enzyme to increase binding affinity for phenylalanine . Interestingly, the variation of either (but not both) of these residues served not only to increase phenylalanine acceptance, but also to enhance overall turnover and ammonia-lyase activity .…”

Section: Structure and Mechanism Of Aromatic Amino Acid Ammonia-lyase...mentioning

confidence: 91%

“…167 Interestingly, the variation of either (but not both) of these residues served not only to increase phenylalanine acceptance, but also to enhance overall turnover and ammonia-lyase activity. 167 This alteration between TAM-and PAL-like activity is reminiscent of studies with PcPAL-Glu484Asn, where decrease in catalytic efficiency with L-phenylalanine and increase with L-tyrosine was observed. 168 These results taken together highlight the importance of fine-tuning mutations for substrate specificity which occur at positions other than the selectivity residues.…”

Section: Arylalanine Aminomutases (Pams and Tams)mentioning

confidence: 98%

“…phenylalanine (unlike TwPAM-Cys107Ser) 253 whilst possessing enantioselectivity superior to that of any other wild-type TAM. 166 Interestingly, OsTAM1 has been shown to have an active site very similar (two amino acid differences) to plant PALs 167 Aside from the engineering of PAMs to perform the TAM reaction, the wild-type substrate scope of the isomerization reaction has been of great interest to researchers in the field of biocatalysis.…”

Section: Synthesis Of β-Arylalaninesmentioning

confidence: 99%

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Synthetic and Therapeutic Applications of Ammonia-lyases and Aminomutases

et al. 2017

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Ammonia-lyases and aminomutases are mechanistically and structurally diverse enzymes which catalyze the deamination and/or isomerization of amino acids in nature by cleaving or shifting a C-N bond. Of the many protein families in which these enzyme activities are found, only a subset have been employed in the synthesis of optically pure fine chemicals or in medical applications. This review covers the natural diversity of these enzymes, highlighting particular enzyme classes that are used within industrial and medical biotechnology. These highlights detail the discovery and mechanistic investigations of these commercially relevant enzymes, along with comparisons of their various applications as stand-alone catalysts, components of artificial biosynthetic pathways and biocatalytic or chemoenzymatic cascades, and therapeutic tools for the potential treatment of various pathologies.

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Section: Structure and Mechanism Of Aromatic Amino Acid Ammonia-lyase...mentioning

confidence: 91%

Section: Arylalanine Aminomutases (Pams and Tams)mentioning

confidence: 98%

Section: Synthesis Of β-Arylalaninesmentioning

confidence: 99%

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Synthetic and Therapeutic Applications of Ammonia-lyases and Aminomutases

et al. 2017

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“…Encouraged by the intrinsic intermolecular transaminase activities exhibited by MIO-PAMs, , we used Tc PAM and (2 S )-styryl-α-alanine to convert trans -3-arylglycidate racemates to arylserines and arylisoserines. In our previous substrate specificity study, we showed that Tc PAM has broad substrate specificity and stereoselectivity toward forming (2 R )- anti -arylserines over their (2 S )- anti -isomers, ranging from 66:34 for 4′-F-phenylserine to 88:12 for 3′–NO 2 –phenylserine .…”

Section: Introductionmentioning

confidence: 99%

“…31 The burst-phase study revealed that the aminated TcPAM (NH 2 −MIO) lifetime was long enough to transfer the amino group intermolecularly from (2S)-styrylα-alanine to various arylacrylates making enantiopure αand βarylamino acids. 21 Encouraged by the intrinsic intermolecular transaminase activities exhibited by MIO-PAMs, 29,32 we used TcPAM and (2S)-styryl-α-alanine to convert trans-3-arylglycidate racemates to arylserines and arylisoserines. In our previous substrate specificity study, we showed that TcPAM has broad substrate specificity and stereoselectivity toward forming (2R)-antiarylserines over their (2S)-anti-isomers, ranging from 66:34 for 4′-F-phenylserine to 88:12 for 3′−NO 2 −phenylserine.…”

Section: ■ Introductionmentioning

confidence: 99%

Intermolecular Amine Transfer to Enantioenriched trans-3-Phenylglycidates by an α/β-Aminomutase to Access Both anti-Phenylserine Isomers

2020

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β-Hydroxy-α-amino acids are noncanonical amino acids with two stereocenters and with useful applications in the pharmaceutical and agrochemical sectors. Here, a 5-methylidene-3,5-dihydro-4H-imidazol-4-one-dependent aminomutase from Taxus canadensis (TcPAM) was repurposed to transfer the amino group irreversibly from (2S)-styryl-α-alanine to exogenously supplied trans-3-phenylglycidate enantiomers, producing antiphenylserines stereoselectively. TcPAM catalysis inverted the intrinsic regioselective chemistry from amination at C β to C α of enantioenriched trans-3-phenylglycidates to make phenylserine predominantly (97%) over phenylisoserine (∼3% relative abundance). Gas chromatography−mass spectrometry analysis of the chiral auxiliary derivatives of the biocatalyzed products confirmed that the amine transfer was stereoselective for each glycidate enantiomer. TcPAM converted (2S,3R)-3-phenylglycidate to (2S)-antiphenylserine predominantly (89%) and (2R,3S)-3-phenylglycidate to (2R)-anti-phenylserine (88%) over their antipodes, with inversion of the configuration at C α in each case. Both glycidate enantiomers formed a small amount (∼10%) of syn-phenylserine by retaining the configuration at C α . The minor syn-isomer likely came from a β-hydroxy oxiranone intermediate formed by intramolecular ring opening of the oxirane ring by the carboxylate before amine transfer. TcPAM had a slight preference toward (2S,3R)-3-phenylglycidate, which was turned over (k cat = 0.3 min −1 ) 1.5 times faster than the (2R,3S)-glycidate (k cat = 0.2 min −1 ). The catalytic efficiencies (k cat app /K M app ≈ 20 M −1 s −1 ) of TcPAM for the antipodes were similar. The kinetic data supported a twosubstrate ping-pong mechanism for the amination of the phenylglycidates, with competitive inhibition at higher glycidate substrate concentrations.

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Understanding Which Residues of the Active Site and Loop Structure of a Tyrosine Aminomutase Define Its Mutase and Lyase Activities

2018

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Site-directed mutations and substrate analogues were used to gain insights into the branch-point reaction of the 3,5-dihydro-5-methylidene-4 H-imidazol-4-one (MIO)-tyrosine aminomutase from Oryza sativa ( OsTAM). Exchanging the active residues of OsTAM (Y125C/N446K) for those in a phenylalanine aminomutase TcPAM altered its substrate specificity from tyrosine to phenylalanine. The aminomutase mechanism of OsTAM surprisingly changed almost exclusively to that of an ammonia lyase making cinnamic acid (>95%) over β-phenylalanine [Walter, T., et al. (2016) Biochemistry 55, 3497-3503]. We hypothesized that the missing electronics or sterics on the aryl ring of the phenylalanine substrate, compared with the sizable electron-donating hydroxyl of the natural tyrosine substrate, influenced the unexpected lyase reactivity of the OsTAM mutant. The double mutant was incubated with 16 α-phenylalanine substituent analogues of varying electronic strengths and sterics. The mutant converted each analogue principally to its acrylate with ∼50% conversion of the p-Br substrate, making only a small amount of the β-amino acid. The inner loop structure over the entrance to the active site was also mutated to assess how the lyase and mutase activities are affected. An OsTAM loop mutant, matching the loop residues of TcPAM, still chiefly made >95% of the acrylate from each substrate. A combined active site:loop mutant was most reactive but remained a lyase, making 10-fold more acrylates than other mutants did. While mutations within the active site changed the substrate specificity of OsTAM, continued exploration is needed to fully understand the interplay among the inner loop, the substrate, and the active site in defining the mutase and lyase activities.

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Mutation of Aryl Binding Pocket Residues Results in an Unexpected Activity Switch in an Oryza sativa Tyrosine Aminomutase

Cited by 3 publications

References 34 publications

Synthetic and Therapeutic Applications of Ammonia-lyases and Aminomutases

Synthetic and Therapeutic Applications of Ammonia-lyases and Aminomutases

Intermolecular Amine Transfer to Enantioenriched trans-3-Phenylglycidates by an α/β-Aminomutase to Access Both anti-Phenylserine Isomers

Understanding Which Residues of the Active Site and Loop Structure of a Tyrosine Aminomutase Define Its Mutase and Lyase Activities

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