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1. Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico

   https://doi.org/10.1038/s41467-020-18619-x  

   Broom, Aron; Rakotoharisoa, Rojo V; Thompson, Michael C; Zarifi, Niayesh; Nguyen, Erin; Mukhametzhanov, Nurzhan; Liu, Lin; Fraser, James S; Chica, Roberto A (December 2020, Nature Communications)

   Abstract The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (k_cat/K_M146 M⁻¹s⁻¹). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (k_cat/K_M103,000 M⁻¹s⁻¹) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data. 

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2. Amino acid interactions that facilitate enzyme catalysis

   https://doi.org/10.1063/5.0041156  

   Coulther, Timothy_A; Ko, Jaeju; Ondrechen, Mary_Jo (May 2021, The Journal of Chemical Physics)

   Interactions in enzymes between catalytic and neighboring amino acids and how these interactions facilitate catalysis are examined. In examples from both natural and designed enzymes, it is shown that increases in catalytic rates may be achieved through elongation of the buffer range of the catalytic residues; such perturbations in the protonation equilibria are, in turn, achieved through enhanced coupling of the protonation equilibria of the active ionizable residues with those of other ionizable residues. The strongest coupling between protonation states for a pair of residues that deprotonate to form an anion (or a pair that accept a proton to form a cation) is achieved when the difference in the intrinsic pKas of the two residues is approximately within 1 pH unit. Thus, catalytic aspartates and glutamates are often coupled to nearby acidic residues. For an anion-forming residue coupled to a cation-forming residue, the elongated buffer range is achieved when the intrinsic pKa of the anion-forming residue is higher than the intrinsic pKa of the (conjugate acid of the) cation-forming residue. Therefore, the high pKa, anion-forming residues tyrosine and cysteine make good coupling partners for catalytic lysine residues. For the anion–cation pairs, the optimum difference in intrinsic pKas is a function of the energy of interaction between the residues. For the energy of interaction ε expressed in units of (ln 10)RT, the optimum difference in intrinsic pKas is within ∼1 pH unit of ε. 

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3. Structural and functional analysis of two SHMT8 variants associated with soybean cyst nematode resistance

   https://doi.org/10.1111/febs.16971  

   Korasick, David A.; Owuocha, Luckio F.; Kandoth, Pramod K.; Tanner, John J.; Mitchum, Melissa G.; Beamer, Lesa J. (October 2023, The FEBS Journal)

   Two amino acid variants in soybean serine hydroxymethyltransferase 8 (SHMT8) are associated with resistance to the soybean cyst nematode (SCN), a devastating agricultural pathogen with worldwide economic impacts on soybean production. SHMT8 is a cytoplasmic enzyme that catalyzes the pyridoxal 5‐phosphate‐dependent conversion of serine and tetrahydrofolate (THF) to glycine and 5,10‐methylenetetrahydrofolate. A previous study of the P130R/N358Y double variant of SHMT8, identified in the SCN‐resistant soybean cultivar (cv.) Forrest, showed profound impairment of folate binding affinity and reduced THF‐dependent enzyme activity, relative to the highly active SHMT8 in cv. Essex, which is susceptible to SCN. Given the importance of SCN‐resistance in soybean agriculture, we report here the biochemical and structural characterization of the P130R and N358Y single variants to elucidate their individual effects on soybean SHMT8. We find that both single variants have reduced THF‐dependent catalytic activity relative to Essex SHMT8 (10‐ to 50‐fold decrease ink_cat/K_m) but are significantly more active than the P130R/N368Y double variant. The kinetic data also show that the single variants lack THF‐substrate inhibition as found in Essex SHMT8, an observation with implications for regulation of the folate cycle. Five crystal structures of the P130R and N358Y variants in complex with various ligands (resolutions from 1.49 to 2.30 Å) reveal distinct structural impacts of the mutations and provide new insights into allosterism. Our results support the notion that the P130R/N358Y double variant in Forrest SHMT8 produces unique and unexpected effects on the enzyme, which cannot be easily predicted from the behavior of the individual variants. 

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4. A Na pump with reduced stoichiometry is up-regulated by brine shrimp in extreme salinities

   https://doi.org/10.1073/pnas.2313999120  

   Artigas, Pablo; Meyer, Dylan J.; Young, Victoria C.; Spontarelli, Kerri; Eastman, Jessica; Strandquist, Evan; Rui, Huan; Roux, Benoît; Birk, Matthew A.; Nakanishi, Hanayo; et al (December 2023, Proceedings of the National Academy of Sciences)

   Brine shrimp (Artemia) are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na⁺,K⁺-ATPase (NKA), a heterodimeric (αβ) pump that usually exports 3Na⁺in exchange for 2 K⁺per hydrolyzed ATP.Artemiaexpress several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2_KK, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines (Xenopusα1’s Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found thatArtemiaexpress two salinity-independent canonical α subunits (α1_NNand α3_NN), as well as two β variants, in addition to the salinity-controlled α2_KK. These β subunits permitted heterologous expression of the α2_KKpump and determination of its CryoEM structure in a closed, ion-free conformation, showing Lys758 residing within the ion-binding cavity. We used electrophysiology to characterize the function of α2_KKpumps and compared it to that ofXenopusα1 (and its α2_KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2_KK-like characteristics toXenopusα1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2_KK’s Lys308 helps to maintain high affinity for external K⁺when Lys758 occupies an ion-binding site. By measuring uptake under voltage clamp of the K⁺-congener⁸⁶Rb⁺, we prove that double-lysine-substituted pumps transport 2Na⁺and 1 K⁺per catalytic cycle. Our results show how the two lysines contribute to generate a pump with reduced stoichiometry allowingArtemiato maintain steeper Na⁺gradients in hypersaline environments. 

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5. Four amino acids define the CO ₂ binding pocket of enoyl-CoA carboxylases/reductases

   https://doi.org/10.1073/pnas.1901471116  

   Stoffel, Gabriele_M M; Saez, David Adrian; DeMirci, Hasan; Vögeli, Bastian; Rao, Yashas; Zarzycki, Jan; Yoshikuni, Yasuo; Wakatsuki, Soichi; Vöhringer-Martinez, Esteban; Erb, Tobias J (July 2019, Proceedings of the National Academy of Sciences)

   Carboxylases are biocatalysts that capture and convert carbon dioxide (CO₂) under mild conditions and atmospheric concentrations at a scale of more than 400 Gt annually. However, how these enzymes bind and control the gaseous CO₂molecule during catalysis is only poorly understood. One of the most efficient classes of carboxylating enzymes are enoyl-CoA carboxylases/reductases (Ecrs), which outcompete the plant enzyme RuBisCO in catalytic efficiency and fidelity by more than an order of magnitude. Here we investigated the interactions of CO₂within the active site of Ecr fromKitasatospora setae. Combining experimental biochemistry, protein crystallography, and advanced computer simulations we show that 4 amino acids, N81, F170, E171, and H365, are required to create a highly efficient CO₂-fixing enzyme. Together, these 4 residues anchor and position the CO₂molecule for the attack by a reactive enolate created during the catalytic cycle. Notably, a highly ordered water molecule plays an important role in an active site that is otherwise carefully shielded from water, which is detrimental to CO₂fixation. Altogether, our study reveals unprecedented molecular details of selective CO₂binding and C–C-bond formation during the catalytic cycle of nature’s most efficient CO₂-fixing enzyme. This knowledge provides the basis for the future development of catalytic frameworks for the capture and conversion of CO₂in biology and chemistry. 

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