Expanding Genetic Code for Protein Lysine and Phenylalanine Modifications

The naturally occurring pyrrolysine (Pyl) incorporation machinery was discovered in methanogenic archaea and some bacteria. In these organisms, Pyl is cotranslationally inserted into proteins and coded by an in-frame UAG codon. Suppression of this UAG codon is mediated by a suppressor tRNA, (tRNA_CUA)^Pyl , that has a CUA anticodon and is acylated with Pyl by pyrrolysyl-tRNA synthetase (PylRS). The PylRS-(tRNA_CUA)^Pyl pair can be directly applied to incorporate Pyl and other lysine derivatives into proteins at amber mutation sites in E. coli and mammalian cells. In the approach of amber codon suppression, evolved PylRSs were selected to synthesize the proteins genetically with lysine and phenylalanine derivatives which contain native or mimic of post-translational modifications (PTMs) or active chemical functional groups for protein labelling and protein folding studies. A photocaged N^6-methyl-L-lysine has been genetically incorporated into proteins at amber codons in Escherichia coli using an evovled PylRS-(tRNA_CUA)^Pyl pair. Its genetic incorporation and following photolysis to recover N?-methyl-L-lysine at phsyiological pH provide a convenient method for the biosythesis of proteins with monomethylated lysines. Using an evolved PylRS-(tRNA_CUA)^Pyl pair, a Se-alkylselenocysteine was genetically incorporated in histone H3. The H3 with mimics of PTMs such as lysine methylation, lysine acetylation, and serine phosphorylation has been synthesized by selective oxidative elimination of Se-alkylselenocysteine and followed Michael addition reactions with different thiol-containing small molecules. Using evolved PylRS -(tRNA_CUA)^Pyl pairs, L-phenylalanine, p-iodo-L-phenylalanine and p-bromo-L-phenylalanine have been genetically incorporated into proteins at amber mutation sites in E. coli. The drastic change of the substrate specificity of PylRS from an aliphatic amino acid to short aromatic amino acids indicates that the PylRS-(tRNA_CUA)^Pyl pair can be evolved for genetic incorporation of a large variety of NAAs into proteins in E. coli. Inspired by the consistent mutations on N346 position, the mutants on N346 and C348 were constructed and evaluated with different L-phenylalanine derivatives. Using PylRS - N346A/C348A (tRNA_CUA)^Pyl pair, more than 30 L-phenylalanine derivatives have been genetically incorporated into proteins at defined sites with amber mutation in E. coli. These breakthroughs and development greatly expand the inventory of genetically encoded NAAs and our abilities to do protein engineering in these cells

Pyrrolysyl-tRNA synthetase variants reveal ancestral aminoacylation function

AbstractPyrrolysyl-tRNA synthetase (PylRS) is a class IIc aminoacyl-tRNA synthetase that is related to phenylalanyl-tRNA synthetase (PheRS). Genetic selection provided PylRS variants with a broad range of specificity for diverse non-canonical amino acids (ncAAs). One variant is a specific phenylalanine-incorporating enzyme. Structural models of the PylRSamino acid complex show that the small pocket size and π-interaction play an important role in specific recognition of Phe and the engineered PylRS active site resembles that of Escherichia coli PheRS

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

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 translationTU Berlin, Open-Access-Mittel – 202

Probing the Catalytic Charge-Relay System in Alanine Racemase with Genetically Encoded Histidine Mimetics

Histidine is a unique amino acid with an imidazole side chain in which both of the nitrogen atoms are capable of serving as a proton donor and proton acceptor in hydrogen bonding interactions. In order to probe the functional role of histidine involved in hydrogen bonding networks, fine-tuning the hydrogen bonding potential of the imidazole side chain is required but not feasible through traditional mutagenesis methods. Here, we show that two close mimetics of histidine, 3-methyl-histidine and thiazole alanine, can be genetically encoded using engineered pyrrolysine incorporation machinery. Replacement of the three histidine residues predicted to be involved in an extended charge-relay system in alanine racemase with 3-methyl-histidine or thiazole alanine shows a dramatic loss in the enzyme’s catalytic efficiency, implying the role of this extended charge-relay system in activating the active site residue Y265, a general acid/base catalyst in the enzyme

Novel Two-Step Process in Cellulose Depolymerization : Hematite-Mediated Photocatalysis by Lytic Polysaccharide Monooxygenase and Fenton Reaction

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A Rationally Designed Pyrrolysyl-tRNA Synthetase Mutant with a Broad Substrate Spectrum

Together with tRNA_CUA^Pyl, a rationally designed pyrrolysyl-tRNA synthetase mutant N346A/C348A has been successfully used for the genetic incorporation of a variety of phenylalanine derivatives with large <i>para</i> substituents into superfolder green fluorescent protein at an amber mutation site in <i>Escherichia coli</i>. This discovery greatly expands the genetically encoded noncanonical amino acid inventory and opens the gate for the genetic incorporation of other phenylalanine derivatives using engineered pyrrolysyl-tRNA synthetase-tRNA_CUA^Pyl pairs

Production of High-Quality Particulate Methane Monooxygenase in High Yields from Methylococcus capsulatus (Bath) with a Hollow-Fiber Membrane Bioreactor

In order to obtain particulate methane monooxygenase (pMMO)-enriched membranes from Methylococcus capsulatus (Bath) with high activity and in high yields, we devised a method to process cell growth in a fermentor adapted with a hollow-fiber bioreactor that allows easy control and quantitative adjustment of the copper ion concentration in NMS medium over the time course of cell culture. This technical improvement in the method for culturing bacterial cells allowed us to study the effects of copper ion concentration in the growth medium on the copper content in the membranes, as well as the specific activity of the enzyme. The optimal copper concentration in the growth medium was found to be 30 to 35 μM. Under these conditions, the pMMO is highly expressed, accounting for 80% of the total cytoplasmic membrane proteins and having a specific activity as high as 88.9 nmol of propylene oxide/min/mg of protein with NADH as the reductant. The copper stoichiometry is ∼13 atoms per pMMO molecule. Analysis of other metal contents provided no evidence of zinc, and only traces of iron were present in the pMMO-enriched membranes. Further purification by membrane solubilization in dodecyl β-d-maltoside followed by fractionation of the protein-detergent complexes according to molecular size by gel filtration chromatography resulted in a good yield of the pMMO-detergent complex and a high level of homogeneity. The pMMO-detergent complex isolated in this way had a molecular mass of 220 kDa and consisted of an αβγ protein monomer encapsulated in a micelle consisting of ca. 240 detergent molecules. The enzyme is a copper protein containing 13.6 mol of copper/mol of pMMO and essentially no iron (ratio of copper to iron, 80:1). Both the detergent-solubilized membranes and the purified pMMO-detergent complex exhibited reasonable, if not excellent, specific activity. Finally, our ability to control the level of expression of the pMMO allowed us to clarify the sensitivity of the enzyme to NADH and duroquinol, the two common reductants used to assay the enzyme

Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase

Pyrrolysyl-tRNA synthetase (PylRS) is a major tool in genetic code expansion with non-canonical amino acids, yet understanding of its structure and activity is incomplete. Here we describe the crystal structure of the previously uncharacterized essential N-terminal domain of this unique enzyme in complex with tRNAPyl. This structure explains why PylRS remains orthogonal in a broad range of organisms, from bacteria to humans. The structure also illustrates why tRNAPyl recognition by PylRS is anticodon-independent; the anticodon does not contact the enzyme. Using standard microbiological culture equipment, we then established a new method for laboratory evolution – a non-continuous counterpart of the previously developed phage-assisted continuous evolution. With this method, we evolved novel PylRS variants with enhanced activity and amino acid specificity. We finally employed an evolved PylRS variant to determine its N-terminal domain structure and show how its mutations improve PylRS activity in the genetic encoding of a non-canonical amino acid

A Facile Method to Synthesize Histones with Posttranslational Modification Mimics

Using an evolved pyrrolysyl-tRNA synthetase-tRNA^Pyl pair, a <i>Se</i>-alkylselenocysteine was genetically incorporated into histone H3 with a high protein expression yield. Quantitative oxidative elimination of <i>Se</i>-alkylselenocysteine followed by Michael addition reactions with various thiol nucleophiles generated biologically active mimics of H3 with posttranslational modifications including lysine methylation, lysine acetylation, and serine phosphorylation