A Novel Glutathione S-Transferase Gtt2 Class (VpGSTT2) Is Found in the Genome of the AHPND/EMS Vibrio parahaemolyticus Shrimp Pathogen.

Glutathione S-transferases are a family of detoxifying enzymes that catalyze the conjugation of reduced glutathione (GSH) with different xenobiotic compounds using either Ser, Tyr, or Cys as a primary catalytic residue. We identified a novel GST in the genome of the shrimp pathogen V. parahaemolyticus FIM- S1708+, a bacterial strain associated with Acute Hepatopancreatic Necrosis Disease (AHPND)/Early Mortality Syndrome (EMS) in cultured shrimp. This new GST class was named Gtt2. It has an atypical catalytic mechanism in which a water molecule instead of Ser, Tyr, or Cys activates the sulfhydryl group of GSH. The biochemical properties of Gtt2 from Vibrio parahaemolyticus (VpGSTT2) were characterized using kinetic and crystallographic methods. Recombinant VpGSTT2 was enzymatically active using GSH and CDNB as substrates, with a specific activity of 5.7 units/mg. Low affinity for substrates was demonstrated using both Michaelis-Menten kinetics and isothermal titration calorimetry. The crystal structure showed a canonical two-domain structure comprising a glutathione binding G-domain and a hydrophobic ligand H domain. A water molecule was hydrogen-bonded to residues Thr9 and Ser 11, as reported for the yeast Gtt2, suggesting a primary role in the reaction. Molecular docking showed that GSH could bind at the G-site in the vicinity of Ser11. G-site mutationsT9A and S11A were analyzed. S11A retained 30% activity, while T9A/S11A showed no detectable activity. VpGSTT2 was the first bacterial Gtt2 characterized, in which residues Ser11 and Thr9 coordinated a water molecule as part of a catalytic mechanism that was characteristic of yeast GTT2. The GTT2 family has been shown to provide protection against metal toxicity; in some cases, excess heavy metals appear in shrimp ponds presenting AHPND/EMS. Further studies may address whether GTT2 in V. parahaemolyticus pathogenic strains may provide a competitive advantage as a novel detoxification mechanism.

Bottom-up synthesis of protein-based nanomaterials from engineered ß-solenoid proteins.

Biomolecular self-assembly is an emerging bottom-up approach for the synthesis of novel nanomaterials. DNA and viruses have both been used to create scaffolds but the former lacks chemical diversity and the latter lack spatial control. To date, the use of protein scaffolds to template materials on the nanoscale has focused on amyloidogenic proteins that are known to form fibrils or two-protein systems where a second protein acts as a cross-linker. We previously developed a unique approach for self-assembly of nanomaterials based on engineering ß-solenoid proteins (BSPs) to polymerize into micrometer-length fibrils. BSPs have highly regular geometries, tunable lengths, and flat surfaces that are amenable to engineering and functionalization. Here, we present a newly engineered BSP based on the antifreeze protein of the beetle Rhagium inquisitor (RiAFP-m9), which polymerizes into stable fibrils under benign conditions. Gold nanoparticles were used to functionalize the RiAFP-m9 fibrils as well as those assembled from the previously described SBAFP-m1 protein. Cysteines incorporated into the sequences provide site-specific gold attachment. Additionally, silver was deposited on the gold-labelled fibrils by electroless plating to create nanowires. These results bolster prospects for programable self-assembly of BSPs to create scaffolds for functional nanomaterials.

RNA binding candidates for human ADAR3 from substrates of a gain of function mutant expressed in neuronal cells.

Human ADAR3 is a catalytically inactive member of the Adenosine Deaminase Acting on RNA (ADAR) protein family, whose active members catalyze A-to-I RNA editing in metazoans. Until now, the reasons for the catalytic incapability of ADAR3 has not been defined and its biological function rarely explored. Yet, its exclusive expression in the brain and involvement in learning and memory suggest a central role in the nervous system. Here we describe the engineering of a catalytically active ADAR3 enzyme using a combination of computational design and functional screening. Five mutations (A389V, V485I, E527Q, Q549R and Q733D) engender RNA deaminase in human ADAR3. By way of its catalytic activity, the ADAR3 pentamutant was used to identify potential binding targets for wild type ADAR3 in a human glioblastoma cell line. Novel ADAR3 binding sites discovered in this manner include the 3'-UTRs of the mRNAs encoding early growth response 1 (EGR1) and dual specificity phosphatase 1 (DUSP1); both known to be activity-dependent immediate early genes that respond to stimuli in the brain. Further studies reveal that the wild type ADAR3 protein can regulate transcript levels for DUSP1 and EGR1, suggesting a novel role ADAR3 may play in brain function.

Carbon Acidity in Enzyme Active Sites.

The pKa values for substrates acting as carbon acids (i.e., C-H deprotonation reactions) in several enzyme active sites are presented. The information needed to calculate them includes the pKa of the active site acid/base catalyst and the equilibrium constant for the deprotonation step. Carbon acidity is obtained from the relation pKeq = p K a r -p K a p = ΔpKa for a proton transfer reaction. Five enzymatic free energy profiles (FEPs) were calculated to obtain the equilibrium constants for proton transfer from carbon in the active site, and six additional proton transfer equilibrium constants were extracted from data available in the literature, allowing substrate C-H pKas to be calculated for 11 enzymes. Active site-bound substrate C-H pKa values range from 5.6 for ketosteroid isomerase to 16 for proline racemase. Compared to values in water, enzymes lower substrate C-H pKas by up to 23 units, corresponding to 31 kcal/mol of carbanion stabilization energy. Calculation of Marcus intrinsic barriers (Δ G 0 ‡ ) for pairs of non-enzymatic/enzymatic reactions shows significant reductions in Δ G 0 ‡ for cofactor-independent enzymes, while pyridoxal phosphate dependent enzymes appear to increase Δ G 0 ‡ to a small extent as a consequence of carbanion resonance stabilization. The large increases in carbon acidity found here are central to the large rate enhancements observed in enzymes that catalyze carbon deprotonation.

In silico stress-strain measurements on self-assembled protein lattices.

Due to their large mechanical strength and potential for functionalization, beta-solenoid proteins show promise as building blocks in biomaterials applications such as two- and three-dimensional scaffolds. We have designed simulation models of two-dimensional square and honeycomb protein lattices by covalently linking a beta-solenoid protein, the spruce budworm antifreeze protein (SBAFP), to symmetric protein multimers. Periodic boundary conditions applied to the simulation cell allow for the simulation of an infinite lattice. We use molecular dynamics to strain the lattice by deforming the simulation cell and measuring the resulting stress tensor. We evaluate the linear portion of stress-strain curves to extract the corresponding bulk and shear elastic moduli. When strained at a rate of 0.3 nm ps-1, the lattices yield a bulk modulus of approximately 3 GPa. This large elastic modulus demonstrates that 2-dimensional structures designed from beta-solenoid proteins can be expected to retain the exceptional material strength of their building blocks.

High Tensile Strength of Engineered ß-Solenoid Fibrils via Sonication and Pulling.

We present estimates of ultimate tensile strength (UTS) for two engineered ß-solenoid protein mutant fibril structures (spruce budworm and Rhagium inquisitor antifreeze proteins) derived from sonication-based measurements and from force pulling molecular dynamics simulations, both in water. Sonication experiments generate limiting scissioned fibrils with a well-defined length-to-width correlation for the mutant spruce budworm protein and the resultant UTS estimate is 0.66 ± 0.08 GPa. For fibrils formed from engineered R. inquisitor antifreeze protein, depending upon geometry, we estimate UTSs of 3.5 ± 3.2-5.5 ± 5.1 GPa for proteins with interfacial disulfide bonds, and 1.6 ± 1.5-2.5 ± 2.3 GPa for the reduced form. The large error bars for the R. inquisitor structures are intrinsic to the broad distribution of limiting scission lengths. Simulations provide pulling velocity-dependent UTSs increasing from 0.2 to 1 GPa in the available speed range, and 1.5 GPa extrapolated to the speeds expected in the sonication experiments. Simulations yield low-velocity values for the Young's modulus of 6.0 GPa. Without protein optimization, these mechanical parameters are similar to those of spider silk and Kevlar, but in contrast to spider silk, these proteins have a precisely known sequence-structure relationship.

Extraordinarily Stable Amyloid Fibrils Engineered from Structurally Defined ß-Solenoid Proteins.

The self-assembly of biological molecules into ordered nanostructures is an attractive method for fabricating novel nanomaterials. Nucleic acid-based nanostructures suffer from limitations to functionalization and stability. Alternatively, protein-based nanostructures have advantageous chemical properties, but design facility lags behind that of nucleic acids. Structurally defined fibrils engineered from ß-solenoid proteins (BSPs) form under mild conditions [Peralta, M. D. R., et al. (2015) ACS Nano 9, 449-463] and are good candidates for novel nanomaterials because of the defined sequence-to-structure relationship and tunable properties. Here, the stability of two types of engineered fibrils was examined using circular dichroism spectroscopy, transmission electron microscopy, and electrophoresis. Both are stable to at least 90 °C, and one survives autoclaving. They are stable toward organic solvents, urea, and pH extremes. One is even stable in 2% sodium dodecyl sulfate with heating. The fibrils show variable resistance to proteolytic digestion: one is resistant to trypsin, but chymotrypsin and proteinase K degrade both. These results show that BSPs have excellent potential for bottom-up design of rugged, functional, amyloid-based nanomaterials.

Site-directed mutant libraries for isolating minimal mutations yielding functional changes.

Powerful, facile new ways to create libraries of site-directed mutants are demonstrated. These include: (1) one-pot-PCR, (2) multi-pot-PCR, and (3) split-mix-PCR. One-pot-PCR uses mutant oligonucleotides to generate megaprimers in situ, and it was used to randomly incorporate 28 mutations in a gabT gene in a single reaction. In more difficult cases, multi-pot-PCR can be employed: mutant megaprimers are synthesized individually, then combined in a single mutagenesis PCR. This method was used to incorporate 14 out of 15 mutations in a pabB gene. Split-mix-PCR is a conceptually novel method for creation of site-directed mutant libraries. Separate PCRs for each mutant primer are performed, followed by pooling the products of the individual reactions. The pooled mixture is re-aliquoted into individual mutant oligonucleotide PCRs. These steps are repeated for each cycle. Split-mix-PCR results in a nearly random distribution of mutation sites, and a distribution of number-of-mutations per gene that is computable and narrow. Split-mix-PCR was applied to the directed evolution of aminodeoxychorismate synthase into anthranilate synthase, and easily allowed the determination of the fewest mutations required for introduction of novel activity.

Biological conversion of gaseous alkenes to liquid chemicals.

Industrial gas-to-liquid (GTL) technologies are well developed. They generally employ syngas, require complex infrastructure, and need high capital investment to be economically viable. Alternatively, biological conversion has the potential to be more efficient, and easily deployed to remote areas on relatively small scales for the utilization of otherwise stranded resources. The present study demonstrates a novel biological GTL process in which engineered Escherichia coli converts C2-C4 gaseous alkenes into liquid diols. Diols are versatile industrially important chemicals, used routinely as antifreeze agents, polymer precursors amongst many other applications. Heterologous co-expression of a monooxygenase and an epoxide hydrolase in E. coli allows whole cell conversion of C2-C4 alkenes for the formation of ethylene glycol, 1,2-propanediol, 1,2-butanediol, and 2,3-butanediol at ambient temperature and pressure in one pot. Increasing intracellular NADH supply via addition of formate and a formate dehydrogenase increases ethylene glycol production titers, resulting in an improved productivity of 9mg/L/h and a final titer of 250mg/L. This represents a novel biological method for GTL conversion of alkenes to industrially valuable diols.

Conversion of aminodeoxychorismate synthase into anthranilate synthase with Janus mutations: mechanism of pyruvate elimination catalyzed by chorismate enzymes.

The central importance of chorismate enzymes in bacteria, fungi, parasites, and plants combined with their absence in mammals makes them attractive targets for antimicrobials and herbicides. Two of these enzymes, anthranilate synthase (AS) and aminodeoxychorismate synthase (ADCS), are structurally and mechanistically similar. The first catalytic step, amination at C2, is common between them, but AS additionally catalyzes pyruvate elimination, aromatizing the aminated intermediate to anthranilate. Despite prior attempts, the conversion of a pyruvate elimination-deficient enzyme into an elimination-proficient one has not been reported. Janus, a bioinformatics method for predicting mutations required to functionally interconvert homologous enzymes, was employed to predict mutations to convert ADCS into AS. A genetic selection on a library of Janus-predicted mutations was performed. Complementation of an AS-deficient strain of Escherichia coli grown on minimal medium led to several ADCS mutants that allow growth in 6 days compared to 2 days for wild-type AS. The purified mutant enzymes catalyze the conversion of chorismate to anthranilate at rates that are ∼50% of the rate of wild-type ADCS-catalyzed conversion of chorismate to aminodeoxychorismate. The residues mutated do not contact the substrate. Molecular dynamics studies suggest that pyruvate elimination is controlled by the conformation of the C2-aminated intermediate. Enzymes that catalyze elimination favor the equatorial conformation, which presents the C2-H to a conserved active site lysine (Lys424) for deprotonation and maximizes stereoelectronic activation. Acid/base catalysis of pyruvate elimination was confirmed in AS and salicylate synthase by showing incorporation of a solvent-derived proton into the pyruvate methyl group and by solvent kinetic isotope effects on pyruvate elimination catalyzed by AS.

Engineering amyloid fibrils from ß-solenoid proteins for biomaterials applications.

Nature provides numerous examples of self-assembly that can potentially be implemented for materials applications. Considerable attention has been given to one-dimensional cross-ß or amyloid structures that can serve as templates for wire growth or strengthen materials such as glue or cement. Here, we demonstrate controlled amyloid self-assembly based on modifications of ß-solenoid proteins. They occur naturally in several contexts (e.g., antifreeze proteins, drug resistance proteins) but do not aggregate in vivo due to capping structures or distortions at their ends. Removal of these capping structures and regularization of the ends of the spruce budworm and rye grass antifreeze proteins yield micron length amyloid fibrils with predictable heights, which can be a platform for biomaterial-based self-assembly. The design process, including all-atom molecular dynamics simulations, purification, and self-assembly procedures are described. Fibril formation with the predicted characteristics is supported by evidence from thioflavin-T fluorescence, circular dichroism, dynamic light scattering, and atomic force microscopy. Additionally, we find evidence for lateral assembly of the modified spruce budworm antifreeze fibrils with sufficient incubation time. The kinetics of polymerization are consistent with those for other amyloid formation reactions and are relatively fast due to the preformed nature of the polymerization nucleus.

Directed evolution of the substrate specificity of dialkylglycine decarboxylase.

Dialkylglycine decarboxylase (DGD) is an unusual pyridoxal phosphate dependent enzyme that catalyzes decarboxylation in the first and transamination in the second half-reaction of its ping-pong catalytic cycle. Directed evolution was employed to alter the substrate specificity of DGD from 2-aminoisobutyrate (AIB) to 1-aminocyclohexane-1-carboxylate (AC6C). Four rounds of directed evolution led to the identification of several mutants, with clones in the final rounds containing five persistent mutations. The best clones show ~2.5-fold decrease in KM and ~2-fold increase in kcat, giving a modest ~5-fold increase in catalytic efficiency for AC6C. Additional rounds of directed evolution did not improve catalytic activity toward AC6C. Only one (S306F) of the five persistent mutations is close to the active site. S306F was observed in all 33 clones except one, and the mutation is shown to stabilize the enzyme toward denaturation. The other four persistent mutations are near the surface of the enzyme. The S306F mutation and the distal mutations all have significant effects on the kinetic parameters for AIB and AC6C. Molecular dynamics simulations suggest that the mutations alter the conformational landscape of the enzyme, favoring a more open active site conformation that facilitates the reactivity of the larger substrate. We speculate that the small increases in kcat/KM for AC6C are due to two constraints. The first is the mechanistic requirement for catalyzing oxidative decarboxylation via a concerted decarboxylation/proton transfer transition state. The second is that DGD must catalyze transamination at the same active site in the second half-reaction of the ping-pong catalytic cycle.

Nitrate reductase (15)N discrimination in Arabidopsis thaliana, Zea mays, Aspergillus niger, Pichea angusta, and Escherichia coli.

Stable (15)N isotopes have been used to examine movement of nitrogen (N) through various pools of the global N cycle. A central reaction in the cycle involves the reduction of nitrate (NO(-) 3) to nitrite (NO(-) 2) catalyzed by nitrate reductase (NR). Discrimination against (15)N by NR is a major determinant of isotopic differences among N pools. Here, we measured in vitro (15)N discrimination by several NRs purified from plants, fungi, and a bacterium to determine the intrinsic (15)N discrimination by the enzyme and to evaluate the validity of measurements made using (15)N-enriched NO(-) 3. Observed NR isotope discrimination ranged from 22 to 32‰ (kinetic isotope effects of 1.022-1.032) among the different isozymes at natural abundance (15)N (0.37%). As the fractional (15)N content of substrate NO(-) 3 increased from natural abundance, the product (15)N fraction deviated significantly from that expected based on substrate enrichment and (15)N discrimination measured at natural abundance. Additionally, isotopic discrimination by denitrifying bacteria used to reduce NO(-) 3 and NO(-) 2 in some protocols became a greater source of error as (15)N enrichment increased. We briefly discuss potential causes of the experimental artifacts with enriched (15)N and recommend against the use of highly enriched (15)N tracers to study N discrimination in plants or soils.

Molecular characterization of novel pyridoxal-5'-phosphate-dependent enzymes from the human microbiome.

Pyridoxal-5'-phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP-dependent enzymes account for ∼1.5% of most prokaryotic genomes and are estimated to be involved in ∼4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP-dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram-positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP-dependent enzyme fold and structure-guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases.

Aspartate aminotransferase: an old dog teaches new tricks.

Aspartate aminotransferase (AAT) is a prototypical pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyzes the reversible interconversion of l-aspartate and α-ketoglutarate with oxalacetate and l-glutamate via a ping-pong catalytic cycle in which the pyridoxamine 5'-phosphate enzyme form is an intermediate. There is a bountiful literature on AAT that spans approximately 60years, and much fundamental mechanistic information on PLP dependent reactions has been gained from its study. Here, we review our recent work on AAT, where we again used it as a test bed for fundamental concepts in PLP chemistry. First, we discuss the role that coenzyme protonation state plays in controlling reaction specificity, then ground state destabilization via hyperconjugation in the external aldimine intermediate is examined. The third topic is light enhancement of catalysis of Cα-H deprotonation by PLP in solution and in AAT, which occurs through a triplet state of the external aldimine intermediate. Lastly, we consider recent advances in our analyses of enzyme multiple sequence alignments for the purpose of predicting mutations that are required to interconvert structurally similar but catalytically distinct enzymes, and the application of our program JANUS to the conversion of AAT into tyrosine aminotransferase.

NMR studies of protonation and hydrogen bond states of internal aldimines of pyridoxal 5'-phosphate acid-base in alanine racemase, aspartate aminotransferase, and poly-L-lysine.

Using (15)N solid-state NMR, we have studied protonation and H-bonded states of the cofactor pyridoxal 5'-phosphate (PLP) linked as an internal aldimine in alanine racemase (AlaR), aspartate aminotransferase (AspAT), and poly-L-lysine. Protonation of the pyridine nitrogen of PLP and the coupled proton transfer from the phenolic oxygen (enolimine form) to the aldimine nitrogen (ketoenamine form) is often considered to be a prerequisite to the initial step (transimination) of the enzyme-catalyzed reaction. Indeed, using (15)N NMR and H-bond correlations in AspAT, we observe a strong aspartate-pyridine nitrogen H-bond with H located on nitrogen. After hydration, this hydrogen bond is maintained. By contrast, in the case of solid lyophilized AlaR, we find that the pyridine nitrogen is neither protonated nor hydrogen bonded to the proximal arginine side chain. However, hydration establishes a weak hydrogen bond to pyridine. To clarify how AlaR is activated, we performed (13)C and (15)N solid-state NMR experiments on isotopically labeled PLP aldimines formed by lyophilization with poly-L-lysine. In the dry solid, only the enolimine tautomer is observed. However, a fast reversible proton transfer involving the ketoenamine tautomer is observed after treatment with either gaseous water or gaseous dry HCl. Hydrolysis requires the action of both water and HCl. The formation of an external aldimine with aspartic acid at pH 9 also produces the ketoenamine form stabilized by interaction with a second aspartic acid, probably via a H-bond to the phenolic oxygen. We postulate that O-protonation is an effectual mechanism for the activation of PLP, as is N-protonation, and that enzymes that are incapable of N-protonation employ this mechanism.

Common enzymological experiments allow free energy profile determination.

The determination of a complete set of rate constants [free energy profiles (FEPs)] for a complex kinetic mechanism is challenging. Enzymologists have devised a variety of informative steady-state kinetic experiments (e.g., Michaelis-Menten kinetics, viscosity dependence of kinetic parameters, kinetic isotope effects, etc.) that each provide distinct information regarding a particular kinetic system. A simple method for combining steady-state experiments in a single analysis is presented here, which allows microscopic rate constants and intrinsic kinetic isotope effects to be determined. It is first shown that Michaelis-Menten kinetic parameters (kcat and Km values), kinetic isotope efffets, solvent viscosity effects, and intermediate partitioning measurements are sufficient to define the rate constants for a reversible uni-uni mechanism with an intermediate, EZ, between the ES and EP complexes. Global optimization provides the framework for combining the independent experimental measurements, and the search for rate constants is performed using algorithms implemented in the biochemical software COPASI. This method is applied to the determination of FEPs for both alanine racemase and triosephosphate isomerase. The FEPs obtained from global optimization agree with those in the literature, with important exceptions. The method opens the door to routine and large-scale determination of FEPs for enzymes.

"On-the-fly" kinetics of enzymatic racemization using deuterium NMR in DNA-based chiral oriented media.

We report the in situ and real-time monitoring of the interconversion of L- and D-alanine-d3 by alanine racemase from Bacillus stearothermophilus directly observed by (2)H NMR spectroscopy in anisotropic phase. The enantiomers are distinguished by the difference of their (2)H quadrupolar splittings in a chiral liquid crystal containing short DNA fragments. The proof-of-principle, the reliability, and the robustness of this new method is demonstrated by the determination of the turnover rates of the enzyme using the Michaelis-Menten model.

Heavy-enzyme kinetic isotope effects on proton transfer in alanine racemase.

The catalytic effects of perdeuterating the pyridoxal phosphate-dependent enzyme alanine racemase from Geobacillus stearothermophilus are reported. The mass of the heavy perdeuterated form is ~5.5% greater than that of the protiated form, causing kinetic isotope effects (KIEs) of ~1.3 on k(cat) and k(cat)/K(M) for both L- and D-alanine. These values increase when Cα-deuterated alanine is used as the substrate. The heavy-enzyme KIEs of ~3 on k(cat)/K(M) with deuterated substrates are greater than the product of the individual heavy-enzyme and primary substrate KIEs. This breakdown of the rule of the geometric mean is likely due to coupled motion between the protein and the proton-transfer reaction coordinate in the rate-limiting step. These data implicate a direct role for protein vibrational motions in barrier crossing for proton-transfer steps in alanine racemase.

Janus: prediction and ranking of mutations required for functional interconversion of enzymes.

Identification of residues responsible for functional specificity in enzymes is a challenging and important problem in protein chemistry. Active-site residues are generally easy to identify, but residues outside the active site are also important to catalysis and their identities and roles are more difficult to determine. We report a method based on analysis of multiple sequence alignments, embodied in our program Janus, for predicting mutations required to interconvert structurally related but functionally distinct enzymes. Conversion of aspartate aminotransferase into tyrosine aminotransferase is demonstrated and compared to previous efforts. Incorporation of 35 predicted mutations resulted in an enzyme with the desired substrate specificity but low catalytic activity. A single round of DNA back-shuffling with wild-type aspartate aminotransferase on this variant generated mutants with tyrosine aminotransferase activities better than those previously realized from rational design or directed evolution. Methods such as this, coupled with computational modeling, may prove invaluable in furthering our understanding of enzyme catalysis and engineering.