Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reaction

Kawamura, Yui; Ishida, Chiharu; Miyata, Ryo; Miyata, Azusa; Hayashi, Seiichiro; Fujinami, Daisuke; Ito, Sohei; Nakano, Shogo

doi:10.1038/s42004-023-01005-1

Cited by 5 publications

(3 citation statements)

References 56 publications

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“…To underscore this, we sought to produce immobilized enzymes for chemical synthesis. We prepared two materials: amine-terminated polystyrene beads and sortagged hyper-thermostable l -amino acid oxidase 2 (HTAncLAAO2), the latter’s C-terminal region modified by LAETGA (Figure A). These materials were conjugated using AcSE5 for 7 days at 4 °C, yielding HTAncLAAO2 immobilized to polystyrene beads.…”

Section: Resultsmentioning

confidence: 99%

Design of Ancestral Sortase E that is Applicable in Protein Biomaterial Synthesis

Miyata,

Chisuga,

Kambe

et al. 2024

ACS Catal.

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Protein biomaterials would have the potential to address global challenges in health and environment. Numerous production methods of the biomaterials exist, with sortase-mediated ligation (SML) being one of the representative technique. SML facilitates the site-specific conjugation of two compounds: donor peptides or proteins with a cell wall sorting signal at their C-terminus and nucleophiles that have oligoglycine at their N-terminal. In our research, we reconstructed an ancestral sortase E, named AcSE5, through a combination of sequence data mining and ancestral sequence reconstruction. AcSE5, a Ca 2+ independent sortase, recognizes donors with LAETG at their Ctermini and can employ both peptides bearing GGG-or GAA-at N-terminus and straight-chained alkylamines as nucleophiles. The enzyme can achieve efficient peptide conjugation, exceeding 70% under optimal conditions. With AcSE5, we synthesized two protein conjugates: Venus-conjugated shark variable new antigen receptor (VNAR) and dual-conjugated VNAR via poly(ethylene) glycol diamine. Direct enzyme immobilization to amino-terminated polystyrene beads was also achieved using AcSE5. The resultant beads, when conjugated with hyper-thermostable ancestral L-amino acid oxidases (HTAncLAAO2), can be employed for deracemization of various racemic amino acids into D-form. For three of phenylalanine derivatives, preparative-scale (100 mg scale) deracemization can be achieved. This process provides high enantiopurity (>99% ee) and isolation yields (>64%) through chemoenzymatic reactions. The immobilized HTAncLAAO2 showed complete resistance to 10 repeated reactions for a total of 240 h. AcSE5 is an excellent enzyme for SML applications.

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Section: Resultsmentioning

confidence: 99%

Design of Ancestral Sortase E that is Applicable in Protein Biomaterial Synthesis

Miyata,

Chisuga,

Kambe

et al. 2024

ACS Catal.

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“…Furthermore, the D-enantiomers of some amino acids are essential in the formation of the bacterial peptidoglycan [17] and are involved in the biosynthesis of natural peptide antibiotics [18]. In this sense, introducing the D-enantiomers of non-canonical amino acids (NCAAs) into the chemistry of living bacterial cells represents an effective approach to substantially enhance the chemical diversity of cellular structures [19–21], but the biocatalysis toolbox for their production is somewhat limited [22,23]. Among the broad group of NCAAs, fluorinated amino acids (FAAs), which contain one or more F atoms, have considerable potential for engineering new chemistries.…”

Section: Introductionmentioning

confidence: 99%

High-yield enzymatic synthesis of mono– and trifluorinated alanine enantiomers

Nieto-Dominguez,

Sako,

Enemark-Rasmussen

et al. 2023

Preprint

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Fluorinated amino acids are a promising entry point for incorporating new-to-Nature chemistries in biological systems. Hence, novel methods are needed for the selective synthesis of these building blocks. In this study, we focused on the enzymatic synthesis of fluorinated alanine enantiomers. To this end, the alanine dehydrogenase fromVibrio proteolyticusand the diaminopimelate dehydrogenase fromSymbiobacterium thermophilumwere applied to thein vitroproduction of (R)-3-fluoroalanine and (S)-3-fluoroalanine, respectively, using 3-fluoropyruvate as the substrate. Additionally, an alanine racemase fromStreptomyces lavendulae, originally selected for setting an alternative enzymatic cascade leading to the production of these non-canonical amino acids, had an unprecedented catalytic efficiency in the β-elimination of fluorine from the monosubstituted fluoroalanine. Thein vitroenzymatic cascade based on the dehydrogenases ofV.proteolyticusandS.thermophilumincluded a cofactor recycling system, whereby a formate dehydrogenase fromPseudomonassp. 101 (either native or engineered) coupled formate oxidation to NAD(P)H formation. Under these conditions, the reaction yields for (R)-3-fluoroalanine and (S)-3-fluoroalanine reached >85% on the fluorinated substrate and proceeded with complete enantiomeric excess. Moreover, the selected dehydrogenases were also able to catalyze the conversion of trifluoropyruvate into trifluorinated alanine, as a first-case example of biocatalysis with amino acids carrying a trifluoromethyl group.

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“…Furthermore, the D-enantiomers of some amino acids are essential in the formation of bacterial peptidoglycans 17 and are involved in the biosynthesis of natural peptide antibiotics 18 . In this sense, introducing the D-enantiomers of noncanonical amino acids (NCAAs) into the chemistry of living bacterial cells represents an effective approach to substantially enhance the chemical diversity of cellular structures [19][20][21] , but the biocatalysis toolbox for their production is somewhat limited 22,23 . Among the broad group of NCAAs, fluorinated amino acids (FAAs), which contain one or more F atoms, have considerable potential for engineering new chemistries.…”

mentioning

confidence: 99%

Enzymatic synthesis of mono- and trifluorinated alanine enantiomers expands the scope of fluorine biocatalysis

Nieto-Domínguez,

Sako,

Enemark-Rasmussen

et al. 2024

Commun Chem

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Fluorinated amino acids serve as an entry point for establishing new-to-Nature chemistries in biological systems, and novel methods are needed for the selective synthesis of these building blocks. In this study, we focused on the enzymatic synthesis of fluorinated alanine enantiomers to expand fluorine biocatalysis. The alanine dehydrogenase from Vibrio proteolyticus and the diaminopimelate dehydrogenase from Symbiobacterium thermophilum were selected for in vitro production of (R)-3-fluoroalanine and (S)-3-fluoroalanine, respectively, using 3-fluoropyruvate as the substrate. Additionally, we discovered that an alanine racemase from Streptomyces lavendulae, originally selected for setting an alternative enzymatic cascade leading to the production of these non-canonical amino acids, had an unprecedented catalytic efficiency in β-elimination of fluorine from the monosubstituted fluoroalanine. The in vitro enzymatic cascade based on the dehydrogenases of V. proteolyticus and S. thermophilum included a cofactor recycling system, whereby a formate dehydrogenase from Pseudomonas sp. 101 (either native or engineered) coupled formate oxidation to NAD(P)H formation. Under these conditions, the reaction yields for (R)-3-fluoroalanine and (S)-3-fluoroalanine reached >85% on the fluorinated substrate and proceeded with complete enantiomeric excess. The selected dehydrogenases also catalyzed the conversion of trifluoropyruvate into trifluorinated alanine as a first-case example of fluorine biocatalysis with amino acids carrying a trifluoromethyl group.

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Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reaction

Cited by 5 publications

References 56 publications

Design of Ancestral Sortase E that is Applicable in Protein Biomaterial Synthesis

Design of Ancestral Sortase E that is Applicable in Protein Biomaterial Synthesis

High-yield enzymatic synthesis of mono– and trifluorinated alanine enantiomers

Enzymatic synthesis of mono- and trifluorinated alanine enantiomers expands the scope of fluorine biocatalysis

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