An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts.

In vitro transcription (IVT) is a DNA-templated process for synthesizing long RNA transcripts, including messenger RNA (mRNA). For many research and commercial applications, IVT of mRNA is typically performed using bacteriophage T7 RNA polymerase (T7 RNAP) owing to its ability to produce full-length RNA transcripts with high fidelity; however, T7 RNAP can also produce immunostimulatory byproducts such as double-stranded RNA that can affect protein expression. Such byproducts require complex purification processes, using methods such as reversed-phase high-performance liquid chromatography, to yield safe and effective mRNA-based medicines. To minimize the need for downstream purification processes, we rationally and computationally engineered a double mutant of T7 RNAP that produces substantially less immunostimulatory RNA during IVT compared with wild-type T7 RNAP. The resulting mutant allows for a simplified production process with similar mRNA potency, lower immunostimulatory content and quicker manufacturing time compared with wild-type T7 RNAP. Herein, we describe the computational design and development of this improved T7 RNAP variant.

Ribonucleotide reductase, a novel drug target for gonorrhea.

Antibiotic-resistant Neisseria gonorrhoeae (Ng) are an emerging public health threat due to increasing numbers of multidrug resistant (MDR) organisms. We identified two novel orally active inhibitors, PTC-847 and PTC-672, that exhibit a narrow spectrum of activity against Ng including MDR isolates. By selecting organisms resistant to the novel inhibitors and sequencing their genomes, we identified a new therapeutic target, the class Ia ribonucleotide reductase (RNR). Resistance mutations in Ng map to the N-terminal cone domain of the α subunit, which we show here is involved in forming an inhibited α4ß4 state in the presence of the ß subunit and allosteric effector dATP. Enzyme assays confirm that PTC-847 and PTC-672 inhibit Ng RNR and reveal that allosteric effector dATP potentiates the inhibitory effect. Oral administration of PTC-672 reduces Ng infection in a mouse model and may have therapeutic potential for treatment of Ng that is resistant to current drugs.

Therapeutic enzyme engineering using a generative neural network.

Enhancing the potency of mRNA therapeutics is an important objective for treating rare diseases, since it may enable lower and less-frequent dosing. Enzyme engineering can increase potency of mRNA therapeutics by improving the expression, half-life, and catalytic efficiency of the mRNA-encoded enzymes. However, sequence space is incomprehensibly vast, and methods to map sequence to function (computationally or experimentally) are inaccurate or time-/labor-intensive. Here, we present a novel, broadly applicable engineering method that combines deep latent variable modelling of sequence co-evolution with automated protein library design and construction to rapidly identify metabolic enzyme variants that are both more thermally stable and more catalytically active. We apply this approach to improve the potency of ornithine transcarbamylase (OTC), a urea cycle enzyme for which loss of catalytic activity causes a rare but serious metabolic disease.

Subunit Interaction Dynamics of Class Ia Ribonucleotide Reductases: In Search of a Robust Assay.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides (NDP) to deoxynucleotides (dNDP), in part, by controlling the ratios and quantities of dNTPs available for DNA replication and repair. The active form of Escherichia coli class Ia RNR is an asymmetric α2ß2 complex in which α2 contains the active site and ß2 contains the stable diferric-tyrosyl radical cofactor responsible for initiating the reduction chemistry. Each dNDP is accompanied by disulfide bond formation. We now report that, under in vitro conditions, ß2 can initiate turnover in α2 catalytically under both "one" turnover (no external reductant, though producing two dCDPs) and multiple turnover (with an external reductant) assay conditions. In the absence of reductant, rapid chemical quench analysis of a reaction of α2, substrate, and effector with variable amounts of ß2 (1-, 10-, and 100-fold less than α2) yields 3 dCDP/α2 at all ratios of α2:ß2 with a rate constant of 8-9 s-1, associated with a rate-limiting conformational change. Stopped-flow fluorescence spectroscopy with a fluorophore-labeled ß reveals that the rate constants for subunit association (163 ± 7 µM-1 s-1) and dissociation (75 ± 10 s-1) are fast relative to turnover, consistent with catalytic ß2. When assaying in the presence of an external reducing system, the turnover number is dictated by the ratio of α2:ß2, their concentrations, and the concentration and nature of the reducing system; the rate-limiting step can change from the conformational gating to a step or steps involving disulfide rereduction, dissociation of the inhibited α4ß4 state, or both. The issues encountered with E. coli RNR are likely of importance in all class I RNRs and are central to understanding the development of screening assays for inhibitors of these enzymes.

Oligonucleotide Sequence Mapping of Large Therapeutic mRNAs via Parallel Ribonuclease Digestions and LC-MS/MS.

Characterization of mRNA sequences is a critical aspect of mRNA drug development and regulatory filing. Herein, we developed a novel bottom-up oligonucleotide sequence mapping workflow combining multiple endonucleases that cleave mRNA at different frequencies. RNase T1, colicin E5, and mazF were applied in parallel to provide complementary sequence coverage for large mRNAs. Combined use of multiple endonucleases resulted in significantly improved sequence coverage: greater than 70% sequence coverage was achieved on mRNAs near 3000 nucleotides long. Oligonucleotide mapping simulations with large human RNA databases demonstrate that the proposed workflow can positively identify a single correct sequence from hundreds of similarly sized sequences. In addition, the workflow is sensitive and specific enough to detect minor sequence impurities such as single nucleotide polymorphisms (SNPs) with a sensitivity of less than 1%. LC-MS/MS-based oligonucleotide sequence mapping can serve as an orthogonal sequence characterization method to techniques such as Sanger sequencing or next-generation sequencing (NGS), providing high-throughput sequence identification and sensitive impurity detection.

Spectroscopic Evidence for a H Bond Network at Y356 Located at the Subunit Interface of Active E. coli Ribonucleotide Reductase.

The reaction catalyzed by E. coli ribonucleotide reductase (RNR) composed of α and ß subunits that form an active α2ß2 complex is a paradigm for proton-coupled electron transfer (PCET) processes in biological transformations. ß2 contains the diferric tyrosyl radical (Y122·) cofactor that initiates radical transfer (RT) over 35 Å via a specific pathway of amino acids (Y122· ⇆ [W48] ⇆ Y356 in ß2 to Y731 ⇆ Y730 ⇆ C439 in α2). Experimental evidence exists for colinear and orthogonal PCET in α2 and ß2, respectively. No mechanistic model yet exists for the PCET across the subunit (α/ß) interface. Here, we report unique EPR spectroscopic features of Y356·-ß, the pathway intermediate generated by the reaction of 2,3,5-F3Y122·-ß2/CDP/ATP with wt-α2, Y731F-α2, or Y730F-α2. High field EPR (94 and 263 GHz) reveals a dramatically perturbed g tensor. [1H] and [2H]-ENDOR reveal two exchangeable H bonds to Y356·: a moderate one almost in-plane with the π-system and a weak one. DFT calculation on small models of Y· indicates that two in-plane, moderate H bonds (rO-H ∼1.8-1.9 Å) are required to reproduce the gx value of Y356· (wt-α2). The results are consistent with a model, in which a cluster of two, almost symmetrically oriented, water molecules provide the two moderate H bonds to Y356· that likely form a hydrogen bond network of water molecules involved in either the reversible PCET across the subunit interface or in H+ release to the solvent during Y356 oxidation.

Glutamate 52-ß at the α/ß subunit interface of Escherichia coli class Ia ribonucleotide reductase is essential for conformational gating of radical transfer.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside diphosphate substrates (S) to deoxynucleotides with allosteric effectors (e) controlling their relative ratios and amounts, crucial for fidelity of DNA replication and repair. Escherichia coli class Ia RNR is composed of α and ß subunits that form a transient, active α2ß2 complex. The E. coli RNR is rate-limited by S/e-dependent conformational change(s) that trigger the radical initiation step through a pathway of 35 Å across the subunit (α/ß) interface. The weak subunit affinity and complex nucleotide-dependent quaternary structures have precluded a molecular understanding of the kinetic gating mechanism(s) of the RNR machinery. Using a docking model of α2ß2 created from X-ray structures of α and ß and conserved residues from a new subclassification of the E. coli Ia RNR (Iag), we identified and investigated four residues at the α/ß interface (Glu350 and Glu52 in ß2 and Arg329 and Arg639 in α2) of potential interest in kinetic gating. Mutation of each residue resulted in loss of activity and with the exception of E52Q-ß2, weakened subunit affinity. An RNR mutant with 2,3,5-trifluorotyrosine radical (F3Y122•) replacing the stable Tyr122• in WT-ß2, a mutation that partly overcomes conformational gating, was placed in the E52Q background. Incubation of this double mutant with His6-α2/S/e resulted in an RNR capable of catalyzing pathway-radical formation (Tyr356•-ß2), 0.5 eq of dCDP/F3Y122•, and formation of an α2ß2 complex that is isolable in pulldown assays over 2 h. Negative stain EM images with S/e (GDP/TTP) revealed the uniformity of the α2ß2 complex formed.

Formal Reduction Potentials of Difluorotyrosine and Trifluorotyrosine Protein Residues: Defining the Thermodynamics of Multistep Radical Transfer.

Redox-active tyrosines (Ys) play essential roles in enzymes involved in primary metabolism including energy transduction and deoxynucleotide production catalyzed by ribonucleotide reductases (RNRs). Thermodynamic characterization of Ys in solution and in proteins remains a challenge due to the high reduction potentials involved and the reactive nature of the radical state. The structurally characterized α3Y model protein has allowed the first determination of formal reduction potentials (E°') for a Y residing within a protein (Berry, B. W.; Martínez-Rivera, M. C.; Tommos, C. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 9739-9743). Using Schultz's technology, a series of fluorotyrosines (FnY, n = 2 or 3) was site-specifically incorporated into α3Y. The global protein properties of the resulting α3(3,5)F2Y, α3(2,3,5)F3Y, α3(2,3)F2Y and α3(2,3,6)F3Y variants are essentially identical to those of α3Y. A protein film square-wave voltammetry approach was developed to successfully obtain reversible voltammograms and E°'s of the very high-potential α3FnY proteins. E°'(pH 5.5; α3FnY(O•/OH)) spans a range of 1040 ± 3 mV to 1200 ± 3 mV versus the normal hydrogen electrode. This is comparable to the potentials of the most oxidizing redox cofactors in nature. The FnY analogues, and the ability to site-specifically incorporate them into any protein of interest, provide new tools for mechanistic studies on redox-active Ys in proteins and on functional and aberrant hole-transfer reactions in metallo-enzymes. The former application is illustrated here by using the determined α3FnY ΔE°'s to model the thermodynamics of radical-transfer reactions in FnY-RNRs and to experimentally test and support the key prediction made.

Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase.

Escherichia coli class Ia ribonucleotide reductase (RNR) is composed of two subunits that form an active α2ß2 complex. The nucleoside diphosphate substrates (NDP) are reduced in α2, 35 Å from the essential diferric-tyrosyl radical (Y122•) cofactor in ß2. The Y122•-mediated oxidation of C439 in α2 occurs by a pathway (Y122 ⇆ [W48] ⇆ Y356 in ß2 to Y731 ⇆ Y730 ⇆ C439 in α2) across the α/ß interface. The absence of an α2ß2 structure precludes insight into the location of Y356 and Y731 at the subunit interface. The proximity in the primary sequence of the conserved E350 to Y356 in ß2 suggested its importance in catalysis and/or conformational gating. To study its function, pH-rate profiles of wild-type ß2/α2 and mutants in which 3,5-difluorotyrosine (F2Y) replaces residue 356, 731, or both are reported in the presence of E350 or E350X (X = A, D, or Q) mutants. With E350, activity is maintained at the pH extremes, suggesting that protonated and deprotonated states of F2Y356 and F2Y731 are active and that radical transport (RT) can occur across the interface by proton-coupled electron transfer at low pH or electron transfer at high pH. With E350X mutants, all RNRs were inactive, suggesting that E350 could be a proton acceptor during oxidation of the interface Ys. To determine if E350 plays a role in conformational gating, the strong oxidants, NO2Y122•-ß2 and 2,3,5-F3Y122•-ß2, were reacted with α2, CDP, and ATP in E350 and E350X backgrounds and the reactions were monitored for pathway radicals by rapid freeze-quench electron paramagnetic resonance spectroscopy. Pathway radicals are generated only when E350 is present, supporting its essential role in gating the conformational change(s) that initiates RT and masking its role as a proton acceptor.

Biophysical Characterization of Fluorotyrosine Probes Site-Specifically Incorporated into Enzymes: E. coli Ribonucleotide Reductase As an Example.

Fluorinated tyrosines (FnY's, n = 2 and 3) have been site-specifically incorporated into E. coli class Ia ribonucleotide reductase (RNR) using the recently evolved M. jannaschii Y-tRNA synthetase/tRNA pair. Class Ia RNRs require four redox active Y's, a stable Y radical (Y·) in the ß subunit (position 122 in E. coli), and three transiently oxidized Y's (356 in ß and 731 and 730 in α) to initiate the radical-dependent nucleotide reduction process. FnY (3,5; 2,3; 2,3,5; and 2,3,6) incorporation in place of Y122-ß and the X-ray structures of each resulting ß with a diferric cluster are reported and compared with wt-ß2 crystallized under the same conditions. The essential diferric-FnY· cofactor is self-assembled from apo FnY-ß2, Fe(2+), and O2 to produce ∼1 Y·/ß2 and ∼3 Fe(3+)/ß2. The FnY· are stable and active in nucleotide reduction with activities that vary from 5% to 85% that of wt-ß2. Each FnY·-ß2 has been characterized by 9 and 130 GHz electron paramagnetic resonance and high-field electron nuclear double resonance spectroscopies. The hyperfine interactions associated with the (19)F nucleus provide unique signatures of each FnY· that are readily distinguishable from unlabeled Y·'s. The variability of the abiotic FnY pKa's (6.4 to 7.8) and reduction potentials (-30 to +130 mV relative to Y at pH 7.5) provide probes of enzymatic reactions proposed to involve Y·'s in catalysis and to investigate the importance and identity of hopping Y·'s within redox active proteins proposed to protect them from uncoupled radical chemistry.

A >200 meV Uphill Thermodynamic Landscape for Radical Transport in Escherichia coli Ribonucleotide Reductase Determined Using Fluorotyrosine-Substituted Enzymes.

Escherichia coli class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y122•) in one subunit (ß2) generates a transient thiyl radical in another subunit (α2) via long-range radical transport (RT) through aromatic amino acid residues (Y122 ⇆ [W48] ⇆ Y356 in ß2 to Y731 ⇆ Y730 ⇆ C439 in α2). Equilibration of Y356•, Y731•, and Y730• was recently observed using site specifically incorporated unnatural tyrosine analogs; however, equilibration between Y122• and Y356• has not been detected. Our recent report of Y356• formation in a kinetically and chemically competent fashion in the reaction of ß2 containing 2,3,5-trifluorotyrosine at Y122 (F3Y122•-ß2) with α2, CDP (substrate), and ATP (effector) has now afforded the opportunity to investigate equilibration of F3Y122• and Y356•. Incubation of F3Y122•-ß2, Y731F-α2 (or Y730F-α2), CDP, and ATP at different temperatures (2-37 °C) provides ΔE°'(F3Y122•-Y356•) of 20 ± 10 mV at 25 °C. The pH dependence of the F3Y122• ⇆ Y356• interconversion (pH 6.8-8.0) reveals that the proton from Y356 is in rapid exchange with solvent, in contrast to the proton from Y122. Insertion of 3,5-difluorotyrosine (F2Y) at Y356 and rapid freeze-quench EPR analysis of its reaction with Y731F-α2, CDP, and ATP at pH 8.2 and 25 °C shows F2Y356• generation by the native Y122•. FnY-RNRs (n = 2 and 3) together provide a model for the thermodynamic landscape of the RT pathway in which the reaction between Y122 and C439 is ∼200 meV uphill.

Reverse Electron Transfer Completes the Catalytic Cycle in a 2,3,5-Trifluorotyrosine-Substituted Ribonucleotide Reductase.

Escherichia coli class Ia ribonucleotide reductase is composed of two subunits (α and ß), which form an α2ß2 complex that catalyzes the conversion of nucleoside 5'-diphosphates to deoxynucleotides (dNDPs). ß2 contains the essential tyrosyl radical (Y122(•)) that generates a thiyl radical (C439(•)) in α2 where dNDPs are made. This oxidation occurs over 35 Å through a pathway of amino acid radical intermediates (Y122 → [W48] → Y356 in ß2 to Y731 → Y730 → C439 in α2). However, chemistry is preceded by a slow protein conformational change(s) that prevents observation of these intermediates. 2,3,5-Trifluorotyrosine site-specifically inserted at position 122 of ß2 (F3Y(•)-ß2) perturbs its conformation and the driving force for radical propagation, while maintaining catalytic activity (1.7 s(-1)). Rapid freeze-quench electron paramagnetic resonance spectroscopy and rapid chemical-quench analysis of the F3Y(•)-ß2, α2, CDP, and ATP (effector) reaction show generation of 0.5 equiv of Y356(•) and 0.5 equiv of dCDP, both at 30 s(-1). In the absence of an external reducing system, Y356(•) reduction occurs concomitant with F3Y reoxidation (0.4 s(-1)) and subsequent to oxidation of all α2s. In the presence of a reducing system, a burst of dCDP (0.4 equiv at 22 s(-1)) is observed prior to steady-state turnover (1.7 s(-1)). The [Y356(•)] does not change, consistent with rate-limiting F3Y reoxidation. The data support a mechanism where Y122(•) is reduced and reoxidized on each turnover and demonstrate for the first time the ability of a pathway radical in an active α2ß2 complex to complete the catalytic cycle.

Formal reduction potential of 3,5-difluorotyrosine in a structured protein: insight into multistep radical transfer.

The reversible Y-O•/Y-OH redox properties of the α3Y model protein allow access to the electrochemical and thermodynamic properties of 3,5-difluorotyrosine. The unnatural amino acid has been incorporated at position 32, the dedicated radical site in α3Y, by in vivo nonsense codon suppression. Incorporation of 3,5-difluorotyrosine gives rise to very minor structural changes in the protein scaffold at pH values below the apparent pK (8.0±0.1) of the unnatural residue. Square-wave voltammetry on α3(3,5)F2Y provides an E°'(Y-O•/Y-OH) of 1026±4 mV versus the normal hydrogen electrode (pH 5.70±0.02) and shows that the fluoro substitutions lower the E°' by -30±3 mV. These results illustrate the utility of combining the optimized α3Y tyrosine radical system with in vivo nonsense codon suppression to obtain the formal reduction potential of an unnatural aromatic residue residing within a well-structured protein. It is further observed that the protein E°' values differ significantly from peak potentials derived from irreversible voltammograms of the corresponding aqueous species. This is notable because solution potentials have been the main thermodynamic data available for amino acid radicals. The findings in this paper are discussed relative to recent mechanistic studies of the multistep radical-transfer process in Escherichia coli ribonucleotide reductase site-specifically labeled with unnatural tyrosine residues.

The Cys319 loop modulates the transition between dehydrogenase and hydrolase conformations in inosine 5'-monophosphate dehydrogenase.

X-ray crystal structures of enzyme-ligand complexes are widely believed to mimic states in the catalytic cycle, but this presumption has seldom been carefully scrutinized. In the case of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase (IMPDH), 10 structures of various enzyme-substrate-inhibitor complexes have been determined. The Cys319 loop is found in at least three different conformations, suggesting that its conformation changes as the catalytic cycle progresses from the dehydrogenase step to the hydrolase reaction. Alternatively, only one conformation of the Cys319 loop may be catalytically relevant while the others are off-pathway. Here we differentiate between these two hypotheses by analyzing the effects of Ala substitutions at three residues of the Cys319 loop, Arg322, Glu323, and Gln324. These mutations have minimal effects on the value of k(cat) (≤5-fold) that obscure large effects (>10-fold) on the microscopic rate constants for individual steps. These substitutions increase the equilibrium constant for the dehydrogenase step but decrease the equilibrium between open and closed conformations of a mobile flap. More dramatic effects are observed when Arg322 is substituted with Glu, which decreases the rates of hydride transfer and hydrolysis by factors of 2000 and 130, respectively. These experiments suggest that the Cys319 loop does indeed have different conformations during the dehydrogenase and hydrolase reactions as suggested by the crystal structures. Importantly, these experiments reveal that the structure of the Cys319 loop modulates the closure of the mobile flap. This conformational change converts the enzyme from a dehydrogenase into hydrolase, suggesting that the conformation of the Cys319 loop may gate the catalytic cycle.