Exploring the potential impact of an expanded genetic code on protein function

With few exceptions, all living organisms encode the same 20 canonical amino acids; however, it remains an open question whether organisms with additional amino acids beyond the common 20 might have an evolutionary advantage. Here, we begin to test that notion by making a large library of mutant enzymes in which 10 structurally distinct noncanonical amino acids were substituted at single sites randomly throughout TEM-1 β-lactamase. A screen for growth on the β-lactam antibiotic cephalexin afforded a unique p-acrylamido-phenylalanine (AcrF) mutation at Val-216 that leads to an increase in catalytic efficiency by increasing kcat, but not significantly affecting KM. To understand the structural basis for this enhanced activity, we solved the X-ray crystal structures of the ligand-free mutant enzyme and of the deacylation-defective wild-type and mutant cephalexin acyl-enzyme intermediates. These structures show that the Val-216–AcrF mutation leads to conformational changes in key active site residues—both in the free enzyme and upon formation of the acyl-enzyme intermediate—that lower the free energy of activation of the substrate transacylation reaction. The functional changes induced by this mutation could not be reproduced by substitution of any of the 20 canonical amino acids for Val-216, indicating that an expanded genetic code may offer novel solutions to proteins as they evolve new activities.

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Structure based protein engineering to confer selectivity of guanine deaminase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331409562x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C437-C437

Author(s):

Aruna Bitra ◽

Ruchi Anand

Keyword(s):

Crystal Structures ◽

Active Site ◽

Catalytic Mechanism ◽

Catalytic Efficiency ◽

Structural Basis ◽

Active Site Residues ◽

X Ray ◽

Secondary Activity ◽

Guanine Deaminase ◽

Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

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Role of the cysteine residues in Arabidopsis thaliana cyclophilin CYP20-3 in peptidyl-prolyl cis–trans isomerase and redox-related functions

Biochemical Journal ◽

10.1042/bj20061092 ◽

2006 ◽

Vol 401 (1) ◽

pp. 287-297 ◽

Cited By ~ 62

Author(s):

Miriam Laxa ◽

Janine König ◽

Karl-Josef Dietz ◽

Andrea Kandlbinder

Keyword(s):

Conformational Changes ◽

Protein Function ◽

Redox Regulation ◽

Catalytic Efficiency ◽

Structural Rearrangement ◽

Disulfide Bridge ◽

Cysteine Residues ◽

Active Centre ◽

Independent Functions

Cyps (cyclophilins) are ubiquitous proteins of the immunophilin superfamily with proposed functions in protein folding, protein degradation, stress response and signal transduction. Conserved cysteine residues further suggest a role in redox regulation. In order to get insight into the conformational change mechanism and functional properties of the chloroplast-located CYP20-3, site-directed mutagenized cysteine→serine variants were generated and analysed for enzymatic and conformational properties under reducing and oxidizing conditions. Compared with the wild-type form, elimination of three out of the four cysteine residues decreased the catalytic efficiency of PPI (peptidyl-prolyl cis–trans isomerase) activity of the reduced CYP20-3, indicating a regulatory role of dithiol–disulfide transitions in protein function. Oxidation was accompanied by conformational changes with a predominant role in the structural rearrangement of the disulfide bridge formed between Cys54 and Cys171. The rather negative Em (midpoint redox potential) of −319 mV places CYP20-3 into the redox hierarchy of the chloroplast, suggesting the activation of CYP20-3 in the light under conditions of limited acceptor availability for photosynthesis as realized under environmental stress. Chloroplast Prx (peroxiredoxins) were identified as interacting partners of CYP20-3 in a DNA-protection assay. A catalytic role in the reduction of 2-Cys PrxA and 2-Cys PrxB was assigned to Cys129 and Cys171. In addition, it was shown that the isomerization and disulfide-reduction activities are two independent functions of CYP20-3 that both are regulated by the redox state of its active centre.

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Protein evolution with an expanded genetic code

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0809543105 ◽

2008 ◽

Vol 105 (46) ◽

pp. 17688-17693 ◽

Cited By ~ 107

Author(s):

Chang C. Liu ◽

Antha V. Mack ◽

Meng-Lin Tsao ◽

Jeremy H. Mills ◽

Hyun Soo Lee ◽

...

Keyword(s):

Amino Acids ◽

Genetic Code ◽

Directed Evolution ◽

Unnatural Amino Acids ◽

Selective Advantage ◽

Display System ◽

Antibody Library ◽

Evolution Of Proteins ◽

Expanded Genetic Code ◽

Selection Of

We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in VH CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded “synthetic” genetic code can confer a selective advantage in the directed evolution of proteins with specific properties.

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Can Power Laws Help Us Understand Gene and Proteome Information?

Advances in Mathematical Physics ◽

10.1155/2013/917153 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

J. A. Tenreiro Machado ◽

António C. Costa ◽

Maria Dulce Quelhas

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Genetic Code ◽

Protein Function ◽

Sequence Data ◽

Proteome Analysis ◽

Power Laws ◽

Linear Sequence ◽

Graphical Visualization

Proteins are biochemical entities consisting of one or more blocks typically folded in a 3D pattern. Each block (a polypeptide) is a single linear sequence of amino acids that are biochemically bonded together. The amino acid sequence in a protein is defined by the sequence of a gene or several genes encoded in the DNA-based genetic code. This genetic code typically uses twenty amino acids, but in certain organisms the genetic code can also include two other amino acids. After linking the amino acids during protein synthesis, each amino acid becomes a residue in a protein, which is then chemically modified, ultimately changing and defining the protein function. In this study, the authors analyze the amino acid sequence using alignment-free methods, aiming to identify structural patterns in sets of proteins and in the proteome, without any other previous assumptions. The paper starts by analyzing amino acid sequence data by means of histograms using fixed length amino acid words (tuples). After creating the initial relative frequency histograms, they are transformed and processed in order to generate quantitative results for information extraction and graphical visualization. Selected samples from two reference datasets are used, and results reveal that the proposed method is able to generate relevant outputs in accordance with current scientific knowledge in domains like protein sequence/proteome analysis.

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The Uncommon Active Site of D-Amino Acid Transaminase from Haliscomenobacter hydrossis: Biochemical and Structural Insights into the New Enzyme

Molecules ◽

10.3390/molecules26165053 ◽

2021 ◽

Vol 26 (16) ◽

pp. 5053

Author(s):

Alina K. Bakunova ◽

Alena Yu. Nikolaeva ◽

Tatiana V. Rakitina ◽

Tatiana Y. Isaikina ◽

Maria G. Khrenova ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Structural Analysis ◽

Active Site ◽

Active Sites ◽

Structural Basis ◽

Active Site Residues ◽

Type Iv ◽

Fold Type ◽

Arginine Residues

Among industrially important pyridoxal-5’-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes.

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Evolution of the Genetic Code by Incorporation of Amino Acids that Improved or Changed Protein Function

Journal of Molecular Evolution ◽

10.1007/s00239-013-9567-y ◽

2013 ◽

Vol 77 (4) ◽

pp. 134-158 ◽

Cited By ~ 21

Author(s):

Brian R. Francis

Keyword(s):

Amino Acids ◽

Genetic Code ◽

Protein Function

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Evolving Bacterial Fitness with an Expanded Genetic Code

10.1101/169409 ◽

2017 ◽

Author(s):

Drew S. Tack ◽

Austin C. Cole ◽

R. Shroff ◽

B.R. Morrow ◽

Andrew D. Ellington

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acid ◽

Noncanonical Amino Acids ◽

Bacterial Fitness ◽

Genetic Code Expansion ◽

Encoding Strategy ◽

Translation Systems ◽

Expanded Genetic Code

AbstractEvolution has for the most part used the canonical 20 amino acids of the natural genetic code to construct proteins. While several theories regarding the evolution of the genetic code have been proposed, experimental exploration of these theories has largely been restricted to phylogenetic and computational modeling. The development of orthogonal translation systems has allowed noncanonical amino acids to be inserted at will into proteins. We have taken advantage of these advances to evolve bacteria to accommodate a 21 amino acid genetic code in which the amber codon ambiguously encodes either 3-nitro-L-tyrosine or stop. Such an ambiguous encoding strategy recapitulates numerous models for genetic code expansion, and we find that evolved lineages first accommodate the unnatural amino acid, and then begin to evolve on a neutral landscape where stop codons begin to appear within genes. The resultant lines represent transitional intermediates on the way to the fixation of a functional 21 amino acid code.

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Structural Basis for Prereceptor Modulation of Plant Hormones by GH3 Proteins

Science ◽

10.1126/science.1221863 ◽

2012 ◽

Vol 336 (6089) ◽

pp. 1708-1711 ◽

Cited By ~ 90

Author(s):

Corey S. Westfall ◽

Chloe Zubieta ◽

Jonathan Herrmann ◽

Ulrike Kapp ◽

Max H. Nanao ◽

...

Keyword(s):

Amino Acids ◽

Conformational Changes ◽

Molecular Basis ◽

Biochemical Analysis ◽

Plant Hormone ◽

Three Dimensional ◽

Structural Basis ◽

Hormone Action ◽

Plant Signaling ◽

Carboxyl Terminal

Acyl acid amido synthetases of the GH3 family act as critical prereceptor modulators of plant hormone action; however, the molecular basis for their hormone selectivity is unclear. Here, we report the crystal structures of benzoate-specific Arabidopsis thaliana AtGH3.12/PBS3 and jasmonic acid–specific AtGH3.11/JAR1. These structures, combined with biochemical analysis, define features for the conjugation of amino acids to diverse acyl acid substrates and highlight the importance of conformational changes in the carboxyl-terminal domain for catalysis. We also identify residues forming the acyl acid binding site across the GH3 family and residues critical for amino acid recognition. Our results demonstrate how a highly adaptable three-dimensional scaffold is used for the evolution of promiscuous activity across an enzyme family for modulation of plant signaling molecules.

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Structural Models for the Dynamic Effects of Loss-of-Function Variants in the Human SIM1 Protein Transcriptional Activation Domain

Biomolecules ◽

10.3390/biom10091314 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1314

Author(s):

Mathew A. Coban ◽

Patrick R. Blackburn ◽

Murray L. Whitelaw ◽

Mieke M. van Haelst ◽

Paldeep S. Atwal ◽

...

Keyword(s):

Transcription Factor ◽

Conformational Changes ◽

Transcriptional Activation ◽

Protein Function ◽

Structural Model ◽

Protein Complexes ◽

Conformational Dynamics ◽

Basic Research ◽

Structural Basis ◽

Loss Of Function

Single-minded homologue 1 (SIM1) is a transcription factor with numerous different physiological and developmental functions. SIM1 is a member of the class I basic helix-loop-helix-PER-ARNT-SIM (bHLH–PAS) transcription factor family, that includes several other conserved proteins, including the hypoxia-inducible factors, aryl hydrocarbon receptor, neuronal PAS proteins, and the CLOCK circadian regulator. Recent studies of HIF-a-ARNT and CLOCK-BMAL1 protein complexes have revealed the organization of their bHLH, PASA, and PASB domains and provided insight into how these heterodimeric protein complexes form; however, experimental structures for SIM1 have been lacking. Here, we describe the first full-length atomic structural model for human SIM1 with its binding partner ARNT in a heterodimeric complex and analyze several pathogenic variants utilizing state-of-the-art simulations and algorithms. Using local and global positional deviation metrics, deductions to the structural basis for the individual mutants are addressed in terms of the deleterious structural reorganizations that could alter protein function. We propose new experiments to probe these hypotheses and examine an interesting SIM1 dynamic behavior. The conformational dynamics demonstrates conformational changes on local and global regions that represent a mechanism for dysfunction in variants presented. In addition, we used our ab initio hybrid model for further prediction of variant hotspots that can be engineered to test for counter variant (restoration of wild-type function) or basic research probe.

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