Reconstruction of Hyper‐Thermostable Ancestral L‐Amino Acid Oxidase to Perform Deracemization to D‐Amino Acids

Abstract

L‐amino acid oxidases (LAAOs) with broad substrate specificity can be used in the deracemization of D,L‐amino acids (D,L‐AAs) to their D‐enantiomers. Hyper‐thermostable LAAO (HTAncLAAO) was designed through a combination of manual sequence data mining and ancestral sequence reconstruction. Soluble expression of HTAncLAAO (>50 mg/L) can be achieved using an E. coli system. HTAncLAAO, which recognizes seven L‐AAs as substrates, exhibits extremely high thermal stability and long‐term stability; the t1/2 value was 95 °C and <5% activity loss after incubation of the enzyme at 30 °C for 1 week. Deracemization could be achieved at 40 °C with a small amount of enzymes compared to previously reported LAAOs. A total of 0.4 mg (2 U) of HTAncLAAO is enough to deracemize three D,L‐AAs at a preparative scale with high enantiomeric excess (>99 % ee, D‐enantiomer). These results suggest that HTAncLAAO is an excellent biocatalyst to perform this deracemization.

Reaction Mechanism of Ancestral l-Lys α-Oxidase from Caulobacter Species Studied by Biochemical, Structural, and Computational Analysis

“…A total of three sequences (Table S1), which were annotated as "hypothetical proteins", were obtained by applying the procedure described in Figure S1. Utilizing the original Python script named "MAFFT2ASR.py" (https: //github.com/shognakano/MAFFT2ASR), 6 an ancestral sequence called AncLLysO2 was generated utilizing the obtained sequences as a template. In MAFFT2ASR.py script, the following processes were automatically performed: 1. multiple sequence alignment by MAFFT, 23 2. generation of a phylogenetic tree by PhyML 24 with the maximum likelihood method, and 3. reconstruction of the ancestral sequence by PAML 25 utilizing the data generated.…”

“…14−16 This is a convenient way to perform the estimation of enzymatic properties by biochemical assay and structure determination using X-ray crystallography. 17 The usefulness of this method for enzyme screening is demonstrated by examples of other successful enzyme discoveries including that of a highly thermostable LAAO for stereoinversion from L-amino acids to D-form, 6 a novel proximity biotinylation enzyme named AirID, 16 and an L-glutamate decarboxylase that can be applied in gram-scale synthesis of γamino butyric acid from L-Glu. 18 In the phylogenetically based screening method, success or failure is strongly dependent on which sequence is used as a template to screen enzymes.…”

“…ASR is a sequence-based protein design method used to infer progenitor sequences located on each node of a phylogenetic tree utilizing multiple sequence alignments and the tree data of protein sequences. , Compared with native enzymes, the generated ancestral enzymes often exhibit outstanding properties, such as high thermal stability and productivity, without sacrificing activity. − This is a convenient way to perform the estimation of enzymatic properties by biochemical assay and structure determination using X-ray crystallography . The usefulness of this method for enzyme screening is demonstrated by examples of other successful enzyme discoveries including that of a highly thermostable LAAO for stereoinversion from l -amino acids to d -form, a novel proximity biotinylation enzyme named AirID, and an l -glutamate decarboxylase that can be applied in gram-scale synthesis of γ-amino butyric acid from l -Glu …”

“…l -Amino acids (L-AAs) are essential biomolecules not only because they are parts of proteins but also because they regulate various physiological functions. L-AA derivatives are small molecular hormones, such as epinephrine, norepinephrine, dopamine, and thyroid hormones, and the production of NO by the metabolism of l -Arg contributes to activation of the mTOR pathway. , The L-AA metabolism regulates physiological functions, and l -amino acid oxidase (LAAO) is one of the most well-known enzymes catalyzing the oxidation of the α-amino group of L-AAs to imino acid. , The products are released into the solvent and hydrolyzed to keto acids. , The reduced FAD, FADH – , is reoxidized by the O 2 molecule, and H 2 O 2 is generated as a second product. , LAAOs are applied to various fields, such as quantification of specific L-AAs and synthesis of fine chemicals. ,− LAAOs primarily belong to the FAD-dependent amine oxidase (FAO) superfamily, which is formed by more than 9000 non-redundant sequences; functionally uncharacterized sequences make up more than 95% of these sequences …”

“… 4 , 5 LAAOs are applied to various fields, such as quantification of specific L-AAs and synthesis of fine chemicals. 3 , 6 − 9 LAAOs primarily belong to the FAD-dependent amine oxidase (FAO) superfamily, which is formed by more than 9000 non-redundant sequences; functionally uncharacterized sequences make up more than 95% of these sequences. 10 …”