Computational-Designed Enzyme for β-Tyrosine Production in Lignin Valorization

Lignin is an underutilized sustainable source of aromatic compounds. To valorize the low-value lignin monomers, we proposed an efficient strategy, involving enzymatic conversion from trans-p-hydroxycinnamic acids to generate valued-added canonical and non-canonical aromatic amino acids. Among them, β-amino acids are recognized as building blocks for bioactive natural products and pharmaceutical ingredients due to their attractive antitumor properties. Using computational enzyme design, the (R)-β-selective phenylalanine aminomutase from Taxus chinensis (TchPAM) was successfully mutated to accept β-tyrosine as the substrate, as well as to generate the (R)-β-tyrosine with excellent enantiopurity (ee > 99%) as the unique product from trans-p-hydroxycinnamic acid. Moreover, the kinetic parameters were determined for the reaction of four Y424 enzyme variants with the synthesis of different phenylalanine and tyrosine enantiomers. In the ammonia elimination reaction of (R)-β-tyrosine, the variants Y424N and Y424C displayed a two-fold increased catalytic efficiency of the wild type. In this work, a binding pocket in the active site, including Y424, K427, I431, and E455, was examined for its influence on the β-enantioselectivity of this enzyme family. Combining the upstream lignin depolymerization and downstream production, a sustainable value chain based on lignin is enabled. In summary, we report a β-tyrosine synthesis process from a monolignol component, offering a new way for lignin valorization by biocatalyst modification.

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Probing the Molecular Determinant for the Catalytic Efficiency of l-Arabinose Isomerase from Bacillus licheniformis

Applied and Environmental Microbiology ◽

10.1128/aem.02254-09 ◽

2010 ◽

Vol 76 (5) ◽

pp. 1653-1660 ◽

Cited By ~ 23

Author(s):

Ponnandy Prabhu ◽

Marimuthu Jeya ◽

Jung-Kul Lee

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Bacillus Licheniformis ◽

Catalytic Efficiency ◽

Homology Model ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Dynamics Simulation ◽

High Turnover ◽

Arabinose Isomerase

ABSTRACT Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.

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In Silico Analysis of β-Galactosidases Primary and Secondary Structure in relation to Temperature Adaptation

Journal of Amino Acids ◽

10.1155/2014/475839 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 10

Author(s):

Vijay Kumar ◽

Nikhil Sharma ◽

Tek Chand Bhalla

Keyword(s):

Amino Acids ◽

Secondary Structure ◽

Aromatic Amino Acids ◽

In Silico Analysis ◽

Catalytic Efficiency ◽

Life Forms ◽

Temperature Tolerance ◽

Temperature Adaptation ◽

Thermal Habitat ◽

Cold Active

β-D-Galactosidases (EC 3.2.1.23) hydrolyze the terminal nonreducing β-D-galactose residues in β-D-galactosides and are ubiquitously present in all life forms including extremophiles. Eighteen microbial β-galactosidase protein sequences, six each from psychrophilic, mesophilic, and thermophilic microbes, were analyzed. Primary structure reveals alanine, glycine, serine, and arginine to be higher in psychrophilic β-galactosidases whereas valine, glutamine, glutamic acid, phenylalanine, threonine, and tyrosine are found to be statistically preferred by thermophilic β-galactosidases. Cold active β-galactosidase has a strong preference towards tiny and small amino acids, whereas high temperature inhabitants had higher content of basic and aromatic amino acids. Thermophilic β-galactosidases have higher percentage of α-helix region responsible for temperature tolerance while cold loving β-galactosidases had higher percentage of sheet and coil region. Secondary structure analysis revealed that charged and aromatic amino acids were significant for sheet region of thermophiles. Alanine was found to be significant and high in the helix region of psychrophiles and valine counters in thermophilic β-galactosidase. Coil region of cold active β-galactosidase has higher content of tiny amino acids which explains their high catalytic efficiency over their counterparts from thermal habitat. The present study has revealed the preference or prevalence of certain amino acids in primary and secondary structure of psychrophilic, mesophilic, and thermophilic β-galactosidase.

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One-Pot Biocatalytic Preparation of Enantiopure Unusual α-Amino Acids from α-Hydroxy Acids via a Hydrogen-Borrowing Dual-Enzyme Cascade

Catalysts ◽

10.3390/catal10121470 ◽

2020 ◽

Vol 10 (12) ◽

pp. 1470

Author(s):

Fei Liu ◽

Junping Zhou ◽

Meijuan Xu ◽

Taowei Yang ◽

Minglong Shao ◽

...

Keyword(s):

Amino Acids ◽

Mandelic Acid ◽

Catalytic Efficiency ◽

Lactobacillus Brevis ◽

Building Blocks ◽

Hydroxy Acids ◽

One Pot ◽

Diverse Range ◽

Enzyme Cascade ◽

Keto Acids

Unusual α-amino acids (UAAs) are important fundamental building blocks and play a key role in medicinal chemistry. Here, we constructed a hydrogen-borrowing dual-enzyme cascade for efficient synthesis of UAAs from α-hydroxy acids (α-HAs). D-mandelate dehydrogenase from Lactobacillus brevis (LbMDH) was screened for the catalysis of α-HAs to α-keto acids but with low activity towards aliphatic α-HAs. Therefore, we rational engineered LbMDH to improve its activity towards aliphatic α-HAs. The substitution of residue Leu243 located in the substrate entrance channel with nonpolar amino acids like Met, Trp, and Ile significantly influenced the enzyme activity towards different α-HAs. Compared with wild type (WT), variant L243W showed 103 U/mg activity towards D-α-hydroxybutyric acid, 1.7 times of the WT’s 60.2 U/mg, while its activity towards D-mandelic acid decreased. Variant L243M showed 2.3 times activity towards D-mandelic acid compared to WT, and its half-life at 40 °C increased to 150.2 h comparing with 98.5 h of WT. By combining LbMDH with L-leucine dehydrogenase from Bacillus cereus, the synthesis of structurally diverse range of UAAs from α-HAs was constructed. We achieved 90.7% conversion for L-phenylglycine production and 66.7% conversion for L-α-aminobutyric acid production. This redox self-sufficient cascade provided high catalytic efficiency and generated pure products.

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Role of transmembrane helix 6 in substrate recognition of the amino acid transporter MhsT

10.1101/2020.04.03.022970 ◽

2020 ◽

Author(s):

Dorota Focht ◽

Caroline Neumann ◽

Joseph Lyons ◽

Ander Eguskiza Bilbao ◽

Rikard Blunck ◽

...

Keyword(s):

Amino Acids ◽

Binding Site ◽

Substrate Recognition ◽

Transmembrane Helix ◽

Aromatic Amino Acids ◽

Binding Pocket ◽

Common Mechanism ◽

Bacillus Halodurans ◽

Recognition Mechanism ◽

Slc6 Family

AbstractMhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na+-dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate-bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly-Met-Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C)ΦG motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity.

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Conformationally rigid aromatic amino acids as potential building blocks for abiotic foldamers

Organic & Biomolecular Chemistry ◽

10.1039/c0ob00593b ◽

2011 ◽

Vol 9 (2) ◽

pp. 367-369 ◽

Cited By ~ 12

Author(s):

Veera V. E. Ramesh ◽

Arup Roy ◽

Kuruppanthara N. Vijayadas ◽

Amol M. Kendhale ◽

Panchami Prabhakaran ◽

...

Keyword(s):

Amino Acids ◽

Aromatic Amino Acids ◽

Building Blocks

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Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids

Molecules ◽

10.3390/molecules25194418 ◽

2020 ◽

Vol 25 (19) ◽

pp. 4418

Author(s):

Hsueh-Wei Tseng ◽

Tobias Baumann ◽

Huan Sun ◽

Yane-Shih Wang ◽

Zoya Ignatova ◽

...

Keyword(s):

Amino Acids ◽

Aromatic Amino Acids ◽

Binding Pocket ◽

Trna Synthetase ◽

Rational Approach ◽

Translation Efficiency ◽

Amino Acid Residues ◽

Trna Synthetases ◽

Methanosarcina Mazei ◽

Genetic Code Expansion

In protein engineering and synthetic biology, Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS), with its cognate tRNAPyl, is one of the most popular tools for site-specific incorporation of non-canonical amino acids (ncAAs). Numerous orthogonal pairs based on engineered MmPylRS variants have been developed during the last decade, enabling a substantial genetic code expansion, mainly with aliphatic pyrrolysine analogs. However, comparatively less progress has been made to expand the substrate range of MmPylRS towards aromatic amino acid residues. Therefore, we set to further expand the substrate scope of orthogonal translation by a semi-rational approach; redesigning the MmPylRS efficiency. Based on the randomization of residues from the binding pocket and tRNA binding domain, we identify three positions (V401, W417 and S193) crucial for ncAA specificity and enzyme activity. Their systematic mutagenesis enabled us to generate MmPylRS variants dedicated to tryptophan (such as β-(1-Azulenyl)-l-alanine or 1-methyl-l-tryptophan) and tyrosine (mainly halogenated) analogs. Moreover, our strategy also significantly improves the orthogonal translation efficiency with the previously activated analog 3-benzothienyl-l-alanine. Our study revealed the engineering of both first shell and distant residues to modify substrate specificity as an important strategy to further expand our ability to discover and recruit new ncAAs for orthogonal translation

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Synthesis and properties of MIDA boronate containing aromatic amino acids: New peptide building blocks

Organic & Biomolecular Chemistry ◽

10.1039/c0ob00847h ◽

2011 ◽

Vol 9 (6) ◽

pp. 1864 ◽

Cited By ~ 13

Author(s):

Neil Colgin ◽

Tony Flinn ◽

Steven L. Cobb

Keyword(s):

Amino Acids ◽

Aromatic Amino Acids ◽

Building Blocks

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Identification and characterization of ace1-type acetylcholinesterase in insecticide-resistant and -susceptible Propylaea japonica (Thunberg)

Bulletin of Entomological Research ◽

10.1017/s0007485317000682 ◽

2017 ◽

Vol 108 (2) ◽

pp. 253-262 ◽

Cited By ~ 2

Author(s):

M.M. Wang ◽

L.Y. Xing ◽

Z.W. Ni ◽

G. Wu

Keyword(s):

Amino Acids ◽

Disulfide Bonds ◽

Aromatic Amino Acids ◽

Binding Pocket ◽

Catalytic Triad ◽

Determining Factors ◽

Omega Loop ◽

Predacious Species ◽

First Time

AbstractCharacterization and gene cloning of acetylecholinesterase (AChE) in the insecticide-resistant (R) and -susceptible (S) insects have been reported in the past. However, the studies focused mostly on herbivorous pests, rather than predacious species, such as ladybird beetles. Using R and S Propylaea japonica (thunberg), a full-length cDNA sequence (2928 bp) of the ace1-type AChE gene was determined for the first time. The ace1 encoding a protein of 645 amino acids contained typical conserved motifs, such as FGESAG domains, catalytic triad, acyl pocket, oxyanino hole, choline binding site, peripheral anionic site, omega loop and conserved aromatic residues. R P. japonica displayed 50-times greater resistance to chlorpyrifos or mathamidophos with a significantly lower AChE sensitivity to paraoxon, malaoxon, chlorpyrifos or methamidophos than its S counterpart. Five amino acids in the ace1 of R P. japonica differed from those found in S P. japonica. One of them, F358S, located in the acyl-binding pocket, might play a crucial role in the resistance of the insect to organophosphates (OPs). Whereas, K493E and I538V, which were close to some of the conserved aromatic amino acids (i.e., H509, Y511, and W499) in the gorge, and G571R and T576A near C593 that formed the disulfide bonds with C471, might also involve in the change of insecticide resistance in P. japonica. AChE insensitivity and amino acid replacements, particularly F358S, might be the determining factors in the alteration of OPs-resistance in P. japonica.

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