Peripheral positions encode transport specificity in the small multidrug resistance exporters.

In secondary active transporters, a relatively limited set of protein folds have evolved diverse solute transport functions. Because of the conformational changes inherent to transport, altering substrate specificity typically involves remodeling the entire structural landscape, limiting our understanding of how novel substrate specificities evolve. In the current work, we examine a structurally minimalist family of model transport proteins, the small multidrug resistance (SMR) transporters, to understand the molecular basis for the emergence of a novel substrate specificity. We engineer a selective SMR protein to promiscuously export quaternary ammonium antiseptics, similar to the activity of a clade of multidrug exporters in this family. Using combinatorial mutagenesis and deep sequencing, we identify the necessary and sufficient molecular determinants of this engineered activity. Using X-ray crystallography, solid-supported membrane electrophysiology, binding assays, and a proteoliposome-based quaternary ammonium antiseptic transport assay that we developed, we dissect the mechanistic contributions of these residues to substrate polyspecificity. We find that substrate preference changes not through modification of the residues that directly interact with the substrate but through mutations peripheral to the binding pocket. Our work provides molecular insight into substrate promiscuity among the SMRs and can be applied to understand multidrug export and the evolution of novel transport functions more generally.

Substrate recruitment via eIF2γ enhances catalytic efficiency of a holophosphatase that terminates the integrated stress response.

Dephosphorylation of pSer51 of the α subunit of translation initiation factor 2 (eIF2αP) terminates signaling in the integrated stress response (ISR). A trimeric mammalian holophosphatase comprised of a protein phosphatase 1 (PP1) catalytic subunit, the conserved C-terminally located ~70 amino acid core of a substrate-specific regulatory subunit (PPP1R15A/GADD34 or PPP1R15B/CReP) and G-actin (an essential cofactor) efficiently dephosphorylate eIF2αP in vitro. Unlike their viral or invertebrate counterparts, with whom they share the conserved 70 residue core, the mammalian PPP1R15s are large proteins of more than 600 residues. Genetic and cellular observations point to a functional role for regions outside the conserved core of mammalian PPP1R15A in dephosphorylating its natural substrate, the eIF2 trimer. We have combined deep learning technology, all-atom molecular dynamics simulations, X-ray crystallography, and biochemistry to uncover binding of the γ subunit of eIF2 to a short helical peptide repeated four times in the functionally important N terminus of human PPP1R15A that extends past its conserved core. Binding entails insertion of Phe and Trp residues that project from one face of an α-helix formed by the conserved repeats of PPP1R15A into a hydrophobic groove exposed on the surface of eIF2γ in the eIF2 trimer. Replacing these conserved Phe and Trp residues with Ala compromises PPP1R15A function in cells and in vitro. These findings suggest mechanisms by which contacts between a distant subunit of eIF2 and elements of PPP1R15A distant to the holophosphatase active site contribute to dephosphorylation of eIF2αP by the core PPP1R15 holophosphatase and to efficient termination of the ISR in mammals.

Approaching the catalytic mechanism of protein lysine methyltransferases by biochemical and simulation techniques.

Protein lysine methyltransferases (PKMTs) transfer up to three methyl groups to the side chains of lysine residues in proteins and fulfill important regulatory functions by controlling protein stability, localization and protein/protein interactions. The methylation reactions are highly regulated, and aberrant methylation of proteins is associated with several types of diseases including neurologic disorders, cardiovascular diseases, and various types of cancer. This review describes novel insights into the catalytic machinery of various PKMTs achieved by the combined application of biochemical experiments and simulation approaches during the last years, focusing on clinically relevant and well-studied enzymes of this group like DOT1L, SMYD1-3, SET7/9, G9a/GLP, SETD2, SUV420H2, NSD1/2, different MLLs and EZH2. Biochemical experiments have unraveled many mechanistic features of PKMTs concerning their substrate and product specificity, processivity and the effects of somatic mutations observed in PKMTs in cancer cells. Structural data additionally provided information about the substrate recognition, enzyme-substrate complex formation, and allowed for simulations of the substrate peptide interaction and mechanism of PKMTs with atomistic resolution by molecular dynamics and hybrid quantum mechanics/molecular mechanics methods. These simulation technologies uncovered important mechanistic details of the PKMT reaction mechanism including the processes responsible for the deprotonation of the target lysine residue, essential conformational changes of the PKMT upon substrate binding, but also rationalized regulatory principles like PKMT autoinhibition. Further developments are discussed that could bring us closer to a mechanistic understanding of catalysis of this important class of enzymes in the near future. The results described here illustrate the power of the investigation of enzyme mechanisms by the combined application of biochemical experiments and simulation technologies.

Linear motif specificity in signaling through p38α and ERK2 mitogen-activated protein kinases.

Mitogen-activated protein kinase (MAPK) cascades are essential for eukaryotic cells to integrate and respond to diverse stimuli. Maintaining specificity in signaling through MAPK networks is key to coupling distinct inputs to appropriate cellular responses. Docking sites-short linear motifs found in MAPK substrates, regulators, and scaffolds-can promote signaling specificity through selective interactions, but how they do so remains unresolved. Here, we screened a proteomic library for sequences interacting with the MAPKs extracellular signal-regulated kinase 2 (ERK2) and p38α, identifying selective and promiscuous docking motifs. Sequences specific for p38α had high net charge and lysine content, and selective binding depended on a pair of acidic residues unique to the p38α docking interface. Finally, we validated a set of full-length proteins harboring docking sites selected in our screens to be authentic MAPK interactors and substrates. This study identifies features that help define MAPK signaling networks and explains how specific docking motifs promote signaling integrity.

Structure and biochemical characterization of l-2-hydroxyglutarate dehydrogenase and its role in the pathogenesis of l-2-hydroxyglutaric aciduria.

l-2-hydroxyglutarate dehydrogenase (L2HGDH) is a mitochondrial membrane-associated metabolic enzyme, which catalyzes the oxidation of l-2-hydroxyglutarate (l-2-HG) to 2-oxoglutarate (2-OG). Mutations in human L2HGDH lead to abnormal accumulation of l-2-HG, which causes a neurometabolic disorder named l-2-hydroxyglutaric aciduria (l-2-HGA). Here, we report the crystal structures of Drosophila melanogaster L2HGDH (dmL2HGDH) in FAD-bound form and in complex with FAD and 2-OG and show that dmL2HGDH exhibits high activity and substrate specificity for l-2-HG. dmL2HGDH consists of an FAD-binding domain and a substrate-binding domain, and the active site is located at the interface of the two domains with 2-OG binding to the re-face of the isoalloxazine moiety of FAD. Mutagenesis and activity assay confirmed the functional roles of key residues involved in the substrate binding and catalytic reaction and showed that most of the mutations of dmL2HGDH equivalent to l-2-HGA-associated mutations of human L2HGDH led to complete loss of the activity. The structural and biochemical data together reveal the molecular basis for the substrate specificity and catalytic mechanism of L2HGDH and provide insights into the functional roles of human L2HGDH mutations in the pathogeneses of l-2-HGA.

Discovery of a class of glycosaminoglycan lyases with ultrabroad substrate spectrum and their substrate structure preferences.

Glycosaminoglycan (GAG) lyases are often strictly substrate specific, and it is especially difficult to simultaneously degrade GAGs with different types of glycosidic bonds. Herein, we found a new class of GAG lyases (GAGases) from different bacteria. These GAGases belong to polysaccharide lyase 35 family and share quite low homology with the identified GAG lyases. The most surprising thing is that GAGases can not only degrade three types of GAGs: hyaluronan, chondroitin sulfate, and heparan sulfate but also even one of them can also degrade alginate. Further investigation of structural preferences revealed that GAGases selectively act on GAG domains composed of non/6-O-/N-sulfated hexosamines and d-glucoronic acids as well as on alginate domains composed of d-mannuronic acids. In addition, GAG lyases were once speculated to have evolved from alginate lyases, but no transitional enzymes have been found. The discovery of GAGases not only broadens the category of GAG lyases, provides new enzymatic tools for the structural and functional studies of GAGs with specific structures, but also provides candidates for the evolution of GAG lyases.

Sugar binding of sodium-glucose cotransporters analyzed by voltage-clamp fluorometry.

Sugar absorption is crucial for life and relies on glucose transporters, including sodium-glucose cotransporters (SGLTs). Although the structure of SGLTs has been resolved, the substrate selectivity of SGLTs across diverse isoforms has not been determined owing to the complex substrate-recognition processes and limited analysis methods. Therefore, this study used voltage-clamp fluorometry (VCF) to explore the substrate-binding affinities of human SGLT1 in Xenopus oocytes. VCF analysis revealed high-affinity binding of D-glucose and D-galactose, which are known transported substrates. D-fructose, which is not a transported substrate, also bound to SGLT1, suggesting potential recognition despite the lack of transport activity. VCF analysis using the T287N mutant of the substrate-binding pocket, which has reduced D-glucose transport capacity, showed that its D-galactose-binding affinity exceeded its D-glucose-binding affinity. This suggests that the change in the VCF signal was due to substrate binding to the binding pocket. Both D-fructose and L-sorbose showed similar binding affinities, indicating that SGLT1 preferentially binds to pyranose-form sugars, including D-fructopyranose. Electrophysiological analysis confirmed that D-fructose binding did not affect the SGLT1 transport function. The significance of the VCF assay lies in its ability to measure sugar-protein interactions in living cells, thereby bridging the gap between structural analyses and functional characterizations of sugar transporters. Our findings also provide insights into SGLT substrate selectivity and the potential for developing medicines with reduced side effects by targeting non-glucose sugars with low bioreactivity.

Specific residues and conformational plasticity define the substrate specificity of short-chain dehydrogenases/reductases.

Short-chain dehydrogenases/reductases (SDRs) are one of the most prevalent enzyme families distributed among the sequenced microorganisms. Despite the presence of a conserved catalytic tetrad and high structural similarity, these enzymes exhibit different substrate specificities. The insufficient knowledge regarding the amino acids underlying substrate specificity hinders the understanding of the SDRs' roles in diverse and significant biological processes. Here, we performed bioinformatic analysis, molecular modeling, and mutagenesis studies to identify the key residues that regulate the substrate specificities of two homologous microbial SDRs (i.e., DesE and KduD). Further, we investigated the impact of altering the physicochemical properties of these amino acids on enzyme activity. Interestingly, molecular dynamics simulations also suggest a critical role of enzyme conformational flexibility in substrate recognition and catalysis. Overall, our findings improve the understanding of microbial SDR substrate specificity and shed light on future rational design of more efficient and effective biocatalysts.

Molecular Insights into the Regulatory Landscape of Protein Kinase C-Related Kinase-2 (PRK2/PKN2) Using Targeted Small Compounds.

The protein kinase C-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the PIF-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.

Fine-tuning an aromatic ring-hydroxylating oxygenase to degrade high molecular weight polycyclic aromatic hydrocarbon.

Rieske nonheme iron aromatic ring-hydroxylating oxygenases (RHOs) play pivotal roles in determining the substrate preferences of polycyclic aromatic hydrocarbon (PAH) degraders. However, their potential to degrade high molecular weight PAHs (HMW-PAHs) has been relatively unexplored. NarA2B2 is an RHO derived from a thermophilic Hydrogenibacillus sp. strain N12. In this study, we have identified four "hotspot" residues (V236, Y300, W316, and L375) that may hinder the catalytic capacity of NarA2B2 when it comes to HMW-PAHs. By employing structure-guided rational enzyme engineering, we successfully modified NarA2B2, resulting in NarA2B2 variants capable of catalyzing the degradation of six different types of HMW-PAHs, including pyrene, fluoranthene, chrysene, benzo[a]anthracene, benzo[b]fluoranthene, and benzo[a]pyrene. Three representative variants, NarA2B2W316I, NarA2B2Y300F-W316I, and NarA2B2V236A-W316I-L375F, not only maintain their abilities to degrade low-molecular-weight PAHs (LMW-PAHs) but also exhibited 2 to 4 times higher degradation efficiency for HMW-PAHs in comparison to another isozyme, NarAaAb. Computational analysis of the NarA2B2 variants predicts that these modifications alter the size and hydrophobicity of the active site pocket making it more suitable for HMW-PAHs. These findings provide a comprehensive understanding of the relationship between three-dimensional structure and functionality, thereby opening up possibilities for designing improved RHOs that can be more effectively used in the bioremediation of PAHs.

Arg-73 of the RNA endonuclease MazF in Salmonella enterica subsp. arizonae contributes to guanine and uracil recognition in the cleavage sequence.

The sequence-specific endoribonuclease MazF is widely conserved among prokaryotes. Approximately 20 different MazF cleavage sequences have been discovered, varying from three to seven nucleotides in length. Although MazFs from various prokaryotes were found, the cleavage sequences of most MazFs are unknown. Here, we characterized the conserved MazF of Salmonella enterica subsp. arizonae (MazF-SEA). Using massive parallel sequencing and fluorometric assays, we revealed that MazF-SEA preferentially cleaves the sequences U∧ACG and U∧ACU (∧ represents cleavage sites). In addition, we predicted the 3D structure of MazF-SEA using AlphaFold2 and aligned it with the crystal structure of RNA-bound Bacillus subtilis MazF to evaluate RNA interactions. We found Arg-73 of MazF-SEA interacts with RNAs containing G and U at the third position from the cleavage sites (U∧ACG and U∧ACU). We then obtained the mutated MazF-SEA R73L protein to evaluate the significance of Arg-73 interaction with RNAs containing G and U at this position. We also used fluorometric and kinetic assays and showed the enzymatic activity of MazF-SEA R73L for the sequence UACG and UACU was significantly decreased. These results suggest Arg-73 is essential for recognizing G and U at the third position from the cleavage sites. This is the first study to our knowledge to identify a single residue responsible for RNA recognition by MazF. Owing to its high specificity and ribosome-independence, MazF is useful for RNA cleavage in vitro. These results will likely contribute to increasing the diversity of MazF specificity and to furthering the application of MazF in RNA engineering.

Cleavage efficiency of the intramembrane protease γ-secretase is reduced by the palmitoylation of a substrate's transmembrane domain.

The intramembrane protease γ-secretase has broad physiological functions, but also contributes to Notch-dependent tumors and Alzheimer's disease. While γ-secretase cleaves numerous membrane proteins, only few nonsubstrates are known. Thus, a fundamental open question is how γ-secretase distinguishes substrates from nonsubstrates and whether sequence-based features or post-translational modifications of membrane proteins contribute to substrate recognition. Using mass spectrometry-based proteomics, we identified several type I membrane proteins with short ectodomains that were inefficiently or not cleaved by γ-secretase, including 'pituitary tumor-transforming gene 1-interacting protein' (PTTG1IP). To analyze the mechanism preventing cleavage of these putative nonsubstrates, we used the validated substrate FN14 as a backbone and replaced its transmembrane domain (TMD), where γ-cleavage occurs, with the one of nonsubstrates. Surprisingly, some nonsubstrate TMDs were efficiently cleaved in the FN14 backbone, demonstrating that a cleavable TMD is necessary, but not sufficient for cleavage by γ-secretase. Cleavage efficiencies varied by up to 200-fold. Other TMDs, including that of PTTG1IP, were still barely cleaved within the FN14 backbone. Pharmacological and mutational experiments revealed that the PTTG1IP TMD is palmitoylated, which prevented cleavage by γ-secretase. We conclude that the TMD sequence of a membrane protein and its palmitoylation can be key factors determining substrate recognition and cleavage efficiency by γ-secretase.

Sequestration from Protease Adaptor Confers Differential Stability to Protease Substrate.

According to the N-end rule, the N-terminal residue of a protein determines its stability. In bacteria, the adaptor ClpS mediates proteolysis by delivering substrates bearing specific N-terminal residues to the protease ClpAP. We now report that the Salmonella adaptor ClpS binds to the N terminus of the regulatory protein PhoP, resulting in PhoP degradation by ClpAP. We establish that the PhoP-activated protein MgtC protects PhoP from degradation by outcompeting ClpS for binding to PhoP. MgtC appears to act exclusively on PhoP, as it did not alter the stability of a different ClpS-dependent ClpAP substrate. Removal of five N-terminal residues rendered PhoP stability independent of both the clpS and mgtC genes. By preserving PhoP protein levels, MgtC enables normal temporal transcription of PhoP-activated genes. The identified mechanism provides a simple means to spare specific substrates from an adaptor-dependent protease.

Deciphering the Clinical Significance and Kinase Functions of GSK3α in Colon Cancer by Proteomics and Phosphoproteomics.

GSK3α and GSK3ß are two GSK3 isoforms with 84% overall identity and 98% identity in their catalytic domains. GSK3ß plays important roles in the pathogenesis of cancer, while GSK3α has long been considered a functionally redundant protein of GSK3ß. Few studies have specifically investigated the functions of GSK3α. In this study, unexpectedly, we found that the expression of GSK3α, but not GSK3ß, was significantly correlated with the overall survival of colon cancer patients in 4 independent cohorts. To decipher the roles of GSK3α in colon cancer, we profiled the phosphorylation substrates of GSK3α and uncovered 156 phosphosites from 130 proteins specifically regulated by GSK3α. A number of these GSK3α-mediated phosphosites have never been reported before or have been incorrectly identified as substrates of GSK3ß. Among them, the levels of HSF1S303p, CANXS583p, MCM2S41p, POGZS425p, SRRM2T983p, and PRPF4BS431p were significantly correlated with the overall survival of colon cancer patients. Further pull-down assays identified 23 proteins, such as THRAP3, BCLAF1, and STAU1, showing strong binding affinity to GSK3α. The interaction between THRAP3 and GSK3α was verified by biochemical experiments. Notably, among the 18 phosphosites of THRAP3, phosphorylation at S248, S253, and S682 is specifically mediated by GSK3α. Mutation of S248 to D (S248D), which mimics the effect of phosphorylation, obviously increased cancer cell migration and the binding affinity to proteins related to DNA damage repair. Collectively, this work not only discloses the specific function of GSK3α as a kinase but also suggests GSK3α as a promising therapeutic target for colon cancer.

Oligomeric Structure of Yeast and Other Invertases Governs Specificity.

Invertases, or ß-fructofuranosidases, are metabolic enzymes widely distributed among plants and microorganisms that hydrolyze sucrose and release fructose from various substrates. Invertase was one of the earliest discovered enzymes, first investigated in the mid-nineteenth century, becoming a classical model used in the primary biochemical studies on protein synthesis, activity, and the secretion of glycoproteins. However, it was not until 20 years ago that a member of this family of enzymes was structurally characterized, showing a bimodular arrangement with a ß-propeller catalytic domain, and a ß-sandwich domain with unknown function. Since then, many studies on related plant and fungal enzymes have revealed them as basically monomeric. By contrast, all yeast enzymes in this family that have been characterized so far have shown sophisticated oligomeric structures mediated by the non-catalytic domain, which is also involved in substrate binding, and how this assembly determines the particular specificity of each enzyme. In this chapter, we will review the available structures of yeast invertases to elucidate the mechanism regulating oligomer formation and compare them with other reported dimeric invertases in which the oligomeric assembly has no apparent functional implications. In addition, recent work on a new family of invertases with absolute specificity for the α-(1,2)-bond of sucrose found in cyanobacteria and plant invertases is highlighted.

Charge neutralization of the active site glutamates does not limit substrate binding and transport by small multidrug resistance transporter EmrE.

EmrE, a small multidrug resistance transporter from Escherichia coli, confers broad-spectrum resistance to polyaromatic cations and quaternary ammonium compounds. Previous transport assays demonstrate that EmrE transports a +1 and a +2 substrate with the same stoichiometry of two protons:one cationic substrate. This suggests that EmrE substrate binding capacity is limited to neutralization of the two essential glutamates, E14A and E14B (one from each subunit in the antiparallel homodimer), in the primary binding site. Here, we explicitly test this hypothesis, since EmrE has repeatedly broken expectations for membrane protein structure and transport mechanism. We previously showed that EmrE can bind a +1 cationic substrate and proton simultaneously, with cationic substrate strongly associated with one E14 residue, whereas the other remains accessible to bind and transport a proton. Here, we demonstrate that EmrE can bind a +2 cation substrate and a proton simultaneously using NMR pH titrations of EmrE saturated with divalent substrates, for a net +1 charge in the transport pore. Furthermore, we find that EmrE can alternate access and transport a +2 substrate and proton at the same time. Together, these results lead us to conclude that E14 charge neutralization does not limit the binding and transport capacity of EmrE.

The Arabidopsis SWEET1 and SWEET2 uniporters recognize similar substrates while differing in subcellular localization.

Sugars Will Eventually be Exported Transporters (SWEETs) are central for sugar allocation in plants. The SWEET family has approximately 20 homologs in most plant genomes, and despite extensive research on their structures and molecular functions, it is still unclear how diverse SWEETs recognize different substrates. Previous work using SweetTrac1, a biosensor constructed by the intramolecular fusion of a conformation-sensitive fluorescent protein in the plasma membrane transporter SWEET1 from Arabidopsis thaliana, identified common features in the transporter's substrates. Here, we report SweetTrac2, a new biosensor based on the Arabidopsis vacuole membrane transporter SWEET2, and use it to explore the substrate specificity of this second protein. Our results show that SWEET1 and SWEET2 recognize similar substrates but some with different affinities. Sequence comparison and mutagenesis analysis support the conclusion that the differences in affinity depend on nonspecific interactions involving previously uncharacterized residues in the substrate-binding pocket. Furthermore, SweetTrac2 can be an effective tool for monitoring sugar transport at vacuolar membranes that would be otherwise challenging to study.

Biochemical investigation of the tryptophan biosynthetic enzyme anthranilate phosphoribosyltransferase in plants.

While mammals require the essential amino acid tryptophan (Trp) in their diet, plants and microorganisms synthesize Trp de novo. The five-step Trp pathway starts with the shikimate pathway product, chorismate. Chorismate is converted to the aromatic compound anthranilate, which is then conjugated to a phosphoribosyl sugar in the second step by anthranilate phosphoribosyltransferase (PAT1). As a single-copy gene in plants, all fixed carbon flux to indole and Trp for protein synthesis, specialized metabolism, and auxin hormone biosynthesis proceeds through PAT1. While bacterial PAT1s have been studied extensively, plant PAT1s have escaped biochemical characterization. Using a structure model, we identified putative active site residues that were variable across plants and kinetically characterized six PAT1s (Arabidopsis thaliana (thale cress), Citrus sinensis (sweet orange), Pistacia vera (pistachio), Juglans regia (English walnut), Selaginella moellendorffii (spike moss), and Physcomitrium patens (spreading earth-moss)). We probed the catalytic efficiency, substrate promiscuity, and regulation of these six enzymes and found that the C. sinensis PAT1 is highly specific for its cognate substrate, anthranilate. Investigations of site-directed mutants of the A. thaliana PAT1 uncovered an active site residue that contributes to promiscuity. While Trp inhibits bacterial PAT1 enzymes, the six plant PAT1s that we tested were not modulated by Trp. Instead, the P. patens PAT1 was inhibited by tyrosine, and the S. moellendorffii PAT1 was inhibited by phenylalanine. This structure-informed biochemical examination identified variations in activity, efficiency, specificity, and enzyme-level regulation across PAT1s from evolutionarily diverse plants.

A broad specificity ß-propeller enzyme from Rhodopseudomonas palustris that hydrolyzes many lactones including γ-valerolactone.

Lactones are prevalent in biological and industrial settings, yet there is a lack of information regarding enzymes used to metabolize these compounds. One compound, γ-valerolactone (GVL), is used as a solvent to dissolve plant cell walls into sugars and aromatic molecules for subsequent microbial conversion to fuels and chemicals. Despite the promise of GVL as a renewable solvent for biomass deconstruction, residual GVL can be toxic to microbial fermentation. Here, we identified a Ca2+-dependent enzyme from Rhodopseudomonas palustris (Rpa3624) and showed that it can hydrolyze aliphatic and aromatic lactones and esters, including GVL. Maximum-likelihood phylogenetic analysis of other related lactonases with experimentally determined substrate preferences shows that Rpa3624 separates by sequence motifs into a subclade with preference for hydrophobic substrates. Additionally, we solved crystal structures of this ß-propeller enzyme separately with either phosphate, an inhibitor, or a mixture of GVL and products to define an active site where calcium-bound water and calcium-bound aspartic and glutamic acid residues make close contact with substrate and product. Our kinetic characterization of WT and mutant enzymes combined with structural insights inform a reaction mechanism that centers around activation of a calcium-bound water molecule promoted by general base catalysis and close contacts with substrate and a potential intermediate. Similarity of Rpa3624 with other ß-propeller lactonases suggests this mechanism may be relevant for other members of this emerging class of versatile catalysts.

Identification of SARS-CoV-2 PLpro and 3CLpro human proteome substrates using substrate phage display coupled with protein network analysis.

Viral proteases play key roles in viral replication, and they also facilitate immune escape by proteolyzing diverse target proteins. Deep profiling of viral protease substrates in host cells is beneficial for understanding viral pathogenesis and for antiviral drug discovery. Here, we utilized substrate phage display coupled with protein network analysis to identify human proteome substrates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteases, including papain-like protease (PLpro) and 3C-like protease (3CLpro). We first performed peptide substrates selection of PLpro and 3CLpro, and we then used the top 24 preferred substrate sequences to identify a total of 290 putative protein substrates. Protein network analysis revealed that the top clusters of PLpro and 3CLpro substrate proteins contain ubiquitin-related proteins and cadherin-related proteins, respectively. We verified that cadherin-6 and cadherin-12 are novel substrates of 3CLpro, and CD177 is a novel substrate of PLpro using in vitro cleavage assays. We thus demonstrated that substrate phage display coupled with protein network analysis is a simple and high throughput method to identify human proteome substrates of SARS-CoV-2 viral proteases for further understanding of virus-host interactions.