Replacing a Cysteine Ligand by Selenocysteine in a [NiFe]-Hydrogenase Unlocks Hydrogen Production Activity and Addresses the Role of Concerted Proton-Coupled Electron Transfer in Electrocatalytic Reversibility.

Hydrogenases catalyze hydrogen/proton interconversion that is normally electrochemically reversible (having minimal overpotential requirement), a special property otherwise almost exclusive to platinum metals. The mechanism of [NiFe]-hydrogenases includes a long-range proton-coupled electron-transfer process involving a specific Ni-coordinated cysteine and the carboxylate of a nearby glutamate. A variant in which this cysteine has been exchanged for selenocysteine displays two distinct changes in electrocatalytic properties, as determined by protein film voltammetry. First, proton reduction, even in the presence of H2 (a strong product inhibitor), is greatly enhanced relative to H2 oxidation: this result parallels a characteristic of natural [NiFeSe]-hydrogenases which are superior H2 production catalysts. Second, an inflection (an S-shaped "twist" in the trace) appears around the formal potential, the small overpotentials introduced in each direction (oxidation and reduction) signaling a departure from electrocatalytic reversibility. Concerted proton-electron transfer offers a lower energy pathway compared to stepwise transfers. Given the much lower proton affinity of Se compared to that of S, the inflection provides compelling evidence that concerted proton-electron transfer is important in determining why [NiFe]-hydrogenases are reversible electrocatalysts.

tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut.

Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G-U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G-C base pairs (G3:C70 and G5:C68).

Enzymes known as PylRS offer the remarkable ability to expand the natural genetic code of a living cell with unnatural amino acids. Currently, over 200 unnatural amino acids can be genetically encoded with the help of PylRS and its partner tRNAPyl, enabling us to endow proteins with novel properties, or regulate protein activity using light or inducible cross-linking. One intriguing feature of PylRS enzymes is their ability to avoid cross-reactivity when two PylRS homologs from different organisms-such as those from the archaea Methanosarcina mazei and Methanomethylophilus alvus-are co-expressed in a single cell. This makes it possible to simultaneously encode two unnatural amino acids in a single protein. This study illuminates the elusive mechanism of PylRS specificity by using cryo-electron microscopy, biochemistry and molecular simulations. The interaction of PylRS from M. alvus with its tRNAPyl is best described as two pieces of a jigsaw puzzle; in which PylRS recognizes the unique shape of its cognate tRNA instead of specific nucleotides in the tRNA sequence like other tRNA-binding enzymes. This finding may streamline the rational design of tools for simultaneous genetic incorporation of multiple unnatural amino acids, thereby facilitating the development of valuable proteins for research, medicine, and biotechnology.

Deciphering the Role of Selenoprotein M.

Selenocysteine (Sec), the 21st amino acid, is structurally similar to cysteine but with a sulfur to selenium replacement. This single change retains many of the chemical properties of cysteine but often with enhanced catalytic and redox activity. Incorporation of Sec into proteins is unique, requiring additional translation factors and multiple steps to insert Sec at stop (UGA) codons. These Sec-containing proteins (selenoproteins) are found in all three domains of life where they often are involved in cellular homeostasis (e.g., reducing reactive oxygen species). The essential role of selenoproteins in humans requires us to maintain appropriate levels of selenium, the precursor for Sec, in our diet. Too much selenium is also problematic due to its toxic effects. Deciphering the role of Sec in selenoproteins is challenging for many reasons, one of which is due to their complicated biosynthesis pathway. However, clever strategies are surfacing to overcome this and facilitate production of selenoproteins. Here, we focus on one of the 25 human selenoproteins, selenoprotein M (SELENOM), which has wide-spread expression throughout our tissues. Its thioredoxin motif suggests oxidoreductase function; however, its mechanism and functional role(s) are still being uncovered. Furthermore, the connection of both high and low expression levels of SELENOM to separate diseases emphasizes the medical application for studying the role of Sec in this protein. In this review, we aim to decipher the role of SELENOM through detailing and connecting current evidence. With multiple proposed functions in diverse tissues, continued research is still necessary to fully unveil the role of SELENOM.

Recoding UAG to selenocysteine in Saccharomyces cerevisiae.

Unique chemical and physical properties are introduced by inserting selenocysteine (Sec) at specific sites within proteins. Recombinant and facile production of eukaryotic selenoproteins would benefit from a yeast expression system; however, the selenoprotein biosynthetic pathway was lost in the evolution of the kingdom Fungi as it diverged from its eukaryotic relatives. Based on our previous development of efficient selenoprotein production in bacteria, we designed a novel Sec biosynthesis pathway in Saccharomyces cerevisiae using Aeromonas salmonicida translation components. S. cerevisiae tRNASer was mutated to resemble A. salmonicida tRNASec to allow recognition by S. cerevisiae seryl-tRNA synthetase as well as A. salmonicida selenocysteine synthase (SelA) and selenophosphate synthetase (SelD). Expression of these Sec pathway components was then combined with metabolic engineering of yeast to enable the production of active methionine sulfate reductase enzyme containing genetically encoded Sec. Our report is the first demonstration that yeast is capable of selenoprotein production by site-specific incorporation of Sec.

Creating Selenocysteine-Specific Reporters Using Inteins.

The selenium moiety in selenocysteine (Sec) imparts enhanced chemical properties to this amino acid and ultimately the protein in which it is inserted. These characteristics are attractive for designing highly active enzymes or extremely stable proteins and studying protein folding or electron transfer, to name a few. There are also 25 human selenoproteins, of which many are essential for our survival. The ability to create or study these selenoproteins is significantly hindered by the inability to easily produce them. Engineering translation has yielded simpler systems to facilitate site-specific insertion of Sec; however, Ser misincorporation remains problematic. Therefore, we have designed two Sec-specific reporters which promote high-throughput screening of Sec translation systems to overcome this barrier. This protocol outlines the workflow to engineer these Sec-specific reporters, with the application to any gene of interest and the ability to transfer this strategy to any organism.

Dual incorporation of non-canonical amino acids enables production of post-translationally modified selenoproteins.

Post-translational modifications (PTMs) can occur on almost all amino acids in eukaryotes as a key mechanism for regulating protein function. The ability to study the role of these modifications in various biological processes requires techniques to modify proteins site-specifically. One strategy for this is genetic code expansion (GCE) in bacteria. The low frequency of post-translational modifications in bacteria makes it a preferred host to study whether the presence of a post-translational modification influences a protein's function. Genetic code expansion employs orthogonal translation systems engineered to incorporate a modified amino acid at a designated protein position. Selenoproteins, proteins containing selenocysteine, are also known to be post-translationally modified. Selenoproteins have essential roles in oxidative stress, immune response, cell maintenance, and skeletal muscle regeneration. Their complicated biosynthesis mechanism has been a hurdle in our understanding of selenoprotein functions. As technologies for selenocysteine insertion have recently improved, we wanted to create a genetic system that would allow the study of post-translational modifications in selenoproteins. By combining genetic code expansion techniques and selenocysteine insertion technologies, we were able to recode stop codons for insertion of N ε-acetyl-l-lysine and selenocysteine, respectively, into multiple proteins. The specificity of these amino acids for their assigned position and the simplicity of reverting the modified amino acid via mutagenesis of the codon sequence demonstrates the capacity of this method to study selenoproteins and the role of their post-translational modifications. Moreover, the evidence that Sec insertion technology can be combined with genetic code expansion tools further expands the chemical biology applications.

The selenocysteine toolbox: A guide to studying the 21st amino acid.

Selenocysteine (Sec), the 21st genetically encoded amino acid, is structurally similar to cysteine (Cys) but with a sulfur to selenium replacement. This small change confers Sec with related chemical properties to Cys but often with enhanced reactivity. In organisms, Sec is present in selenoproteins taking on various roles such as cellular maintenance, immune response, hormone regulation, and oxidative stress. The detailed reactions of Sec in these functions remains unclear and has been a difficult question to answer. This is related to the low natural expression of selenoproteins and their complicated biosynthesis pathway. As a result, the focus in selenoprotein research has been on the expansion of tools and techniques to promote research in this area. Over the past two decades there has been immense progress in the development of selenoprotein expression systems, Sec-detection methods, and genomic databases. In this review we have compiled these tools systematically, highlighting their strengths and clarifying the limitations, as a resource for future selenoprotein research.

Diversification of aminoacyl-tRNA synthetase activities via genomic duplication.

Intricate evolutionary events enabled the emergence of the full set of aminoacyl-tRNA synthetase (aaRS) families that define the genetic code. The diversification of aaRSs has continued in organisms from all domains of life, yielding aaRSs with unique characteristics as well as aaRS-like proteins with innovative functions outside translation. Recent bioinformatic analyses have revealed the extensive occurrence and phylogenetic diversity of aaRS gene duplication involving every synthetase family. However, only a fraction of these duplicated genes has been characterized, leaving many with biological functions yet to be discovered. Here we discuss how genomic duplication is associated with the occurrence of novel aaRSs and aaRS-like proteins that provide adaptive advantages to their hosts. We illustrate the variety of activities that have evolved from the primordial aaRS catalytic sites. This precedent underscores the need to investigate currently unexplored aaRS genomic duplications as they may hold a key to the discovery of exciting biological processes, new drug targets, important bioactive molecules, and tools for synthetic biology applications.

Unconventional genetic code systems in archaea.

Archaea constitute the third domain of life, distinct from bacteria and eukaryotes given their ability to tolerate extreme environments. To survive these harsh conditions, certain archaeal lineages possess unique genetic code systems to encode either selenocysteine or pyrrolysine, rare amino acids not found in all organisms. Furthermore, archaea utilize alternate tRNA-dependent pathways to biosynthesize and incorporate members of the 20 canonical amino acids. Recent discoveries of new archaeal species have revealed the co-occurrence of these genetic code systems within a single lineage. This review discusses the diverse genetic code systems of archaea, while detailing the associated biochemical elements and molecular mechanisms.

Uncovering translation roadblocks during the development of a synthetic tRNA.

Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNAUTu1, a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNAUTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.

Continued expansion of the genetic code has required the use of synthetic tRNAs for decoding. Some of these synthetic tRNAs have unique structural features that are not observed in canonical tRNAs. Here, the authors applied single-molecule, biochemical and structural methods to determine whether these distinct features were deleterious for efficient protein translation on the ribosome. With a focus on selenocysteine insertion, the authors explored an allo-tRNA with a 9/3 acceptor domain. They observed a translational roadblock that occurred in A to P site tRNA translocation. This block was mediated by a tertiary interaction across the tRNA core, directing the variable arm position into an unfavorable conformation. A single-nucleotide mutation disrupted this interaction, providing flexibility in the variable arm and promoting efficient protein production.

Using selenocysteine-specific reporters to screen for efficient tRNASec variants.

The unique properties of selenocysteine (Sec) have generated an interest in the scientific community to site-specifically incorporate Sec into a protein of choice. Current technologies have rewired the natural Sec-specific translation factor-dependent selenoprotein biosynthesis pathway by harnessing the canonical elongation factor (EF-Tu) to simplify the requirements for Sec incorporation in Escherichia coli. This strategy is versatile and can be applied to Sec incorporation at any position in a protein of interest. However, selenoprotein production is still limited by yield and serine misincorporation. This protocol outlines a method in E. coli to design and optimize tRNA libraries which can be selected and screened for by the use of Sec-specific intein-based reporters. This provides a fast and simple way to engineer tRNAs with enhanced Sec-incorporation ability.

Harnessing selenocysteine to enhance microbial cell factories for hydrogen production.

Hydrogen is a clean, renewable energy source, that when combined with oxygen, produces heat and electricity with only water vapor as a biproduct. Furthermore, it has the highest energy content by weight of all known fuels. As a result, various strategies have engineered methods to produce hydrogen efficiently and in quantities that are of interest to the economy. To approach the notion of producing hydrogen from a biological perspective, we take our attention to hydrogenases which are naturally produced in microbes. These organisms have the machinery to produce hydrogen, which when cleverly engineered, could be useful in cell factories resulting in large production of hydrogen. Not all hydrogenases are efficient at hydrogen production, and those that are, tend to be oxygen sensitive. Therefore, we provide a new perspective on introducing selenocysteine, a highly reactive proteinogenic amino acid, as a strategy towards engineering hydrogenases with enhanced hydrogen production, or increased oxygen tolerance.

Intein-based Design Expands Diversity of Selenocysteine Reporters.

The presence of selenocysteine in a protein confers many unique properties that make the production of recombinant selenoproteins desirable. Targeted incorporation of Sec into a protein of choice is possible by exploiting elongation factor Tu-dependent reassignment of UAG codons, a strategy that has been continuously improved by a variety of means. Improving selenoprotein yield by directed evolution requires selection and screening markers that are titratable, have a high dynamic range, enable high-throughput screening, and can discriminate against nonspecific UAG decoding. Current screening techniques are limited to a handful of reporters where a cysteine (Cys) or Sec residue normally affords activity. Unfortunately, these existing Cys/Sec-dependent reporters lack the dynamic range of more ubiquitous reporters or suffer from other limitations. Here we present a versatile strategy to adapt established reporters for specific Sec incorporation. Inteins are intervening polypeptides that splice themselves from the precursor protein in an autocatalytic splicing reaction. Using an intein that relies exclusively on Sec for splicing, we show that this intein cassette can be placed in-frame within selection and screening markers, affording reporter activity only upon successful intein splicing. Furthermore, because functional splicing can only occur when a catalytic Sec is present, the amount of synthesized reporter directly measures UAG-directed Sec incorporation. Importantly, we show that results obtained with intein-containing reporters are comparable to the Sec incorporation levels determined by mass spectrometry of isolated recombinant selenoproteins. This result validates the use of these intein-containing reporters to screen for evolved components of a translation system yielding increased selenoprotein amounts.

Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor.

The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. KEY POINTS: • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality.

Selective cysteine-to-selenocysteine changes in a [NiFe]-hydrogenase confirm a special position for catalysis and oxygen tolerance.

In [NiFe]-hydrogenases, the active-site Ni is coordinated by four cysteine-S ligands (Cys; C), two of which are bridging to the Fe(CO)(CN)2 fragment. Substitution of a single Cys residue by selenocysteine (Sec; U) occurs occasionally in nature. Using a recent method for site-specific Sec incorporation into proteins, each of the four Ni-coordinating cysteine residues in the oxygen-tolerant Escherichia coli [NiFe]-hydrogenase-1 (Hyd-1) has been replaced by U to identify its importance for enzyme function. Steady-state solution activity of each Sec-substituted enzyme (on a per-milligram basis) is lowered, although this may reflect the unquantified presence of recalcitrant inactive/immature/misfolded forms. Protein film electrochemistry, however, reveals detailed kinetic data that are independent of absolute activities. Like native Hyd-1, the variants have low apparent KMH2 values, do not produce H2 at pH 6, and display the same onset overpotential for H2 oxidation. Mechanistically important differences were identified for the C576U variant bearing the equivalent replacement found in native [NiFeSe]-hydrogenases, its extreme O2 tolerance (apparent KMH2 and Vmax [solution] values relative to native Hyd-1 of 0.13 and 0.04, respectively) implying the importance of a selenium atom in the position cis to the site where exogenous ligands (H-, H2, O2) bind. Observation of the same unusual electrocatalytic signature seen earlier for the proton transfer-defective E28Q variant highlights the direct role of the chalcogen atom (S/Se) at position 576 close to E28, with the caveat that Se is less effective than S in facilitating proton transfer away from the Ni during H2 oxidation by this enzyme.

Introducing Selenocysteine into Recombinant Proteins in Escherichia coli.

Selenoproteins contain the 21st amino acid, selenocysteine. Selenocysteine is the only amino acid that is synthesized on its cognate tRNA, and it is inserted at specific recoded UGA stop codons via a complex translation system. Although highly similar to cysteine, selenocysteine has unique properties, including a stronger nucleophilic ability and lower reduction potential. Efforts to site-specifically incorporate selenocysteine to create recombinant selenoproteins involve a recoded UAG stop codon and expression of the necessary selenocysteine translation machinery. This article presents a protocol for expressing and purifying selenoproteins in Escherichia coli. © 2021 Wiley Periodicals LLC. Basic Protocol: Recombinant selenoprotein production in E. coli using a rewired translation system.

Naturally Occurring tRNAs With Non-canonical Structures.

Transfer RNA (tRNA) is the central molecule in genetically encoded protein synthesis. Most tRNA species were found to be very similar in structure: the well-known cloverleaf secondary structure and L-shaped tertiary structure. Furthermore, the length of the acceptor arm, T-arm, and anticodon arm were found to be closely conserved. Later research discovered naturally occurring, active tRNAs that did not fit the established 'canonical' tRNA structure. This review discusses the non-canonical structures of some well-characterized natural tRNA species and describes how these structures relate to their role in translation. Additionally, we highlight some newly discovered tRNAs in which the structure-function relationship is not yet fully understood.

Structural and Hydrodynamic Characterization of Dimeric Human Oligoadenylate Synthetase 2.

Oligoadenylate synthetases (OASs) are a family of interferon-inducible enzymes that require double-stranded RNA (dsRNA) as a cofactor. Upon binding dsRNA, OAS undergoes a conformational change and is activated to polymerize ATP into 2'-5'-oligoadenylate chains. The OAS family consists of several isozymes, with unique domain organizations to potentially interact with dsRNA of variable length, providing diversity in viral RNA recognition. In addition, oligomerization of OAS isozymes, potentially OAS1 and OAS2, is hypothesized to be important for 2'-5'-oligoadenylate chain building. In this study, we present the solution conformation of dimeric human OAS2 using an integrated approach involving small-angle x-ray scattering, analytical ultracentrifugation, and dynamic light scattering techniques. We also demonstrate OAS2 dimerization using immunoprecipitation approaches in human cells. Whereas mutation of a key active-site aspartic acid residue prevents OAS2 activity, a C-terminal mutation previously hypothesized to disrupt OAS self-association had only a minor effect on OAS2 activity. Finally, we also present the solution structure of OAS1 monomer and dimer, comparing their hydrodynamic properties with OAS2. In summary, our work presents the first, to our knowledge, dimeric structural models of OAS2 that enhance our understanding of the oligomerization and catalytic function of OAS enzymes.

Hijacking Translation Initiation for Synthetic Biology.

Genetic code expansion (GCE) has revolutionized the field of protein chemistry. Over the past several decades more than 150 different noncanonical amino acids (ncAAs) have been co-translationally installed into proteins within various host organisms. The vast majority of these ncAAs have been incorporated between the start and stop codons within an open reading frame. This requires that the ncAA be able to form a peptide bond at the α-amine, limiting the types of molecules that can be genetically encoded. In contrast, the α-amine of the initiating amino acid is not required for peptide bond formation. Therefore, including the initiator position in GCE allows for co-translational insertion of more diverse molecules that are modified, or completely lacking an α-amine. This review explores various methods which have been used to initiate protein synthesis with diverse molecules both in vitro and in vivo.

Engineering aminoacyl-tRNA synthetases for use in synthetic biology.

Within the broad field of synthetic biology, genetic code expansion (GCE) techniques enable creation of proteins with an expanded set of amino acids. This may be invaluable for applications in therapeutics, bioremediation, and biocatalysis. Central to GCE are aminoacyl-tRNA synthetases (aaRSs) as they link a non-canonical amino acid (ncAA) to their cognate tRNA, allowing ncAA incorporation into proteins on the ribosome. The ncAA-acylating aaRSs and their tRNAs should not cross-react with 20 natural aaRSs and tRNAs in the host, i.e., they need to function as an orthogonal translating system. All current orthogonal aaRS•tRNA pairs have been engineered from naturally occurring molecules to change the aaRS's amino acid specificity or assign the tRNA to a liberated codon of choice. Here we discuss the importance of orthogonality in GCE, laboratory techniques employed to create designer aaRSs and tRNAs, and provide an overview of orthogonal aaRS•tRNA pairs for GCE purposes.