Biochemistry

The glutaminase (GLS) isoforms KGA and GAC are expressed in neurons where they hydrolyze glutamine to produce the excitatory neurotransmitter glutamate. Two de novo gain-of-function mutants of GLS, S482C and H461L, were recently identified in patients with developmental delay, epilepsy, and infantile cataract. These patients exhibited high glutamate and low glutamine concentrations in the brain, suggesting that the GLS mutants have abnormal enzymology. Here, we examined the enzymatic properties of these GLS mutants and found that they exhibit a total (S482C) or partial (H461L) loss of glutamate product inhibition, lifting this restriction on glutamate accumulation. The mutant enzymes also no longer require the anionic activator phosphate to stimulate enzymatic activity or induce filament formation. Structural analysis of the S482C GAC mutant shows the mutation shifts the key catalytic residue Y466 into the catalytically competent position and disrupts a key hydrogen bond between it and the glutamate product, explaining how the S482C mutant has enzymatic activity in the absence of phosphate and is insensitive to glutamate product inhibition. These results shed new light on the mechanism of phosphate activation and glutamate product inhibition of GLS and show that loss of these enzymatic properties disrupts glutamate homeostasis in the brain and causes neurological disease.

When bacterial ribosomes are assembled in vitro, manipulation of incubation temperature and magnesium ion concentration have been an essential procedure, which is a crucial step for the assembly of active large subunits. The present study tackles with this issue to develop a single-step procedure, which can be performed in a physiological condition. We found that GTPase factors EngA and ObgE can complement the changes in temperature and magnesium ion concentrations. In the presence of these factors, ribosome assembly can proceed under physiological conditions, with magnesium ion concentrations below 10 mM, potassium concentrations around 100 mM, and temperatures of 37 {degrees}C. Both the ribosome assembly and translation processes were successfully integrated in the reconstituted cell-free protein synthesis system. Furthermore, we found that these GTPase factors can reassemble the ribosomes to an active state, whose structure was disrupted by EDTA chelation of magnesium ions, indicating that these two factors can reversibly induce the ribosome structure to an intact state. The findings are essential for the bottom-up construction of synthetic cells.

As an ATP-dependent protease, the quality control functions of Lon have been extensively studied and reviewed in the literature. By contrast, very little research has been conducted to investigate Lons physiological functions and its mechanism as a regulatory protease. In this manuscript, we provided a survey of literature and data to convey that the lambda N ({lambda}N) protein is a suitable Escherichia coli Lon (ELon) substrate for studying the role played by Lon in regulating an RNA transcription process. For proof of principle, we demonstrated that the minimal component of the RNA transcription complex containing RNA polymerase (RNAP) and the {sigma} factor can inhibit {lambda}N degradation by ELon through SDS-PAGE, and the carboxyl-terminal of {lambda}N is important for Lon competing with RNAP interaction. Using negative stain electron microscopy, we obtained structural evidence to show that {lambda}N lacking the carboxyl-terminal flanked by residues 99-107 interacted with ELon differently than full-length {lambda}N. Taken together, the activity and EM data provide a starting point for performing a physiological enzymology study on the contribution of ELon toward RNA transcription.

Adenylate kinases (AKs) are phosphotransferases that are frequently employed as models to investigate protein structure-function relationships. Prior studies have shown that AK homologs of different stabilities retain cellular activity in cells following circular permutation that split the AMP binding domain into fragments coded at different ends of the primary structure, such that this domain was no longer embedded as a continuous polypeptide within the core domain. Herein, we show mesophilic and thermophilic AKs having this topological restructuring retain activity and substrate-binding characteristics of the parental AK. While permutation decreased the activity of both AK homologs at physiological temperatures, the catalytic activity of the thermophilic AK increased upon permutation when assayed >30{degrees}C below the melting temperature of the native AK. The thermostabilities of the permuted AKs were uniformly lower than native AKs, and they exhibited multi-phasic unfolding transitions, unlike the native AKs, which presented cooperative thermal unfolding. In addition, proteolytic digestion revealed that permutation destabilized each AK, and mass spectrometry suggested that the new termini within the AMP binding domain were responsible for the increased proteolysis sensitivity. These findings illustrate how changes in contact order can be used to tune enzyme activity and alter folding dynamics in multidomain enzymes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/564053v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1ee9ecdorg.highwire.dtl.DTLVardef@fbb415org.highwire.dtl.DTLVardef@ebdd90org.highwire.dtl.DTLVardef@11f4271_HPS_FORMAT_FIGEXP M_FIG C_FIG

Under conditions of oxidative stress or iron starvation, iron-sulfur cluster biogenesis in E. coli is initiated by the cysteine desulfurase, SufS, via the SUF pathway. SufS is a type II cysteine desulfurase that catalyzes the PLP-dependent breakage of an L-cysteine C-S bond to generate L-alanine and a covalent active site persulfide as products. The persulfide is transferred from SufS to SufE and then to the SufBC2D complex, which utilizes it in iron-sulfur cluster biogenesis. Several lines of evidence suggest two conserved arginine residues that line the solvent side of the SufS active site could be important for function. To investigate the mechanistic roles of R56 and R359, the residues were substituted using site-directed mutagenesis to obtain R56A/K and R359A/K SufS variants. Steady state kinetics indicated R56 and R359 have moderate defects in the desulfurase half reaction but major defects in the transpersulfurase step. Fluorescence polarization binding assays showed that the loss of activity was not due to a defect in forming the SufS/SufE complex. Structural characterization of R56A SufS shows loss of electron density for the 3-4 loop at the R56/G57 positions, consistent with a requirement of R56 for proper loop conformation. The structure of R359A SufS exhibits a conformational change in the 3-4 loop allowing R56 to enter the active site and mimics the residues position in the PLP-cysteine aldimine structure. Taken together, the kinetic, binding, and structural data support a mechanism where R359 plays a role in linking SufS catalysis with modulation of the 3-4 loop to promote a close-approach interaction of SufS and SufE conducive to persulfide transfer.

Toxin-antitoxin (TA) systems, including YoeB-YefM, are important mediators of bacterial physiological changes. Agrobacterium tumefaciens YoeB and YefM are similar to that from E. coli, and interact as a tight heterotetramer with a KD of 653 pM. We have verified that AtYoeB can perform both ribosome-dependent and -independent RNA cleavage. We have also characterized a newly described metal-dependent and pH-sensitive DNA cleaving ability. We note that this DNA cleaving ability is observed at toxin concentrations as low as 150 nM. The dose-dependence of in vitro ribosome-independent RNA and metal-dependent DNA cleavage is equivalent, and requires a ten-fold increase in toxin concentration as opposed to in the presence of the ribosome. The toxin concentration inside bacterial cells is unknown and according to current models, should increase upon activation of YoeB through degradation of the YefM antitoxin. The discovery of general nuclease activity by AtYoeB, and perhaps other YoeB toxins, offers an opportunity to explore the plasticity of this protein fold and its potential role in the evolution of nucleases.

The surface of gram-positive bacteria is a highly regulated environment with specific attachment of proteins required for viability. Sortase enzymes are cysteine transpeptidases that recognize and ligate substrates to the peptidoglycan layer in these microorganisms, which can be highly pathogenic (e.g., Staphylococcus aureus, Streptococcus pyogenes, etc.). As such, sortases represent a potentially novel target for antibiotic development. In addition, the catalytic activity of sortase enzymes is utilized in sortase-mediated ligation (SML) engineering approaches for a variety of uses. In SML experiments, engineered variants of Staphylococcus aureus sortase A (saSrtA) are the most widely used enzymes. One of the mutated amino acids in the previously engineered pentamutant (or saSrtA5M) enzyme is P94. Structural analyses of experimental saSrtA structures revealed that P94 interacts directly with Y187 when saSrtA is in its inactive conformation. While saSrtA5M, developed via directed evolution, contains a P94R mutation, we wanted to interrogate this position further and ask if other single P94 mutations may reveal a greater effect on activity and/or substrate specificity. We created 18 P94X mutations (excluding P94C), and tested relative activity using a fluorescence resonance energy transfer (FRET) assay for 4 substrate sequences: LPATG, LPETG, LPKTG, and LPSTG. We identified several P94 variants that outperformed the single mutant P94R for all peptides tested, including P94A, P94D, P94E, P94G, P94H, P94N, P94Q, P94S, and P94T. We further observed that the reactivity of substrates with variations in the central position of the pentapeptide recognition motif (LPXTG) can be sensitive to the identity of the P94X residue. We tested P94A and P94D saSrtA5M variants and found that, depending on LPXTG sequence, these variants could outperform saSrtA5M in activity > 3-fold. Finally, we compared saSrtA5M and P94D saSrtA5M in a model sortase-mediated ligation reaction using a LPKTG substrate and saw [~]2-fold greater product formation. Taken together, we characterized an important position that modulates substrate access and activity in saSrtA. Furthermore, we argue that future studies which combine rational design and high throughput approaches, e.g., directed evolution, may result in sortase variants with increased SML potential.

Gram-negative bacteria are intrinsically resistant to many antibiotics in part due to the asymmetric architecture and barrier function of their outer membrane (OM). To establish proper lipid asymmetry, cells need to ensure an intricate balance of constituent OM components, especially lipids. In this regard, the conserved, trans-envelope Tol-Pal complex plays a primary role in maintaining OM lipid homeostasis, thus OM integrity, possibly via retrograde phospholipid transport. However, mechanistic details for this process are unknown, owing to the lack of evidence for direct lipid binding. In this study, we discover that the periplasmic protein TolB, a key component of the Tol-Pal system, associates directly with membranes in vitro, via specific interactions with cardiolipin (CL). Using coarse-grained molecular dynamics simulations, we identify a CL-binding site on TolB; a single amino acid mutation at this site abolishes in vitro membrane interaction, consequently impairing cellular Tol-Pal function in maintaining OM homeostasis in Escherichia coli. Curiously, we find that the functional requirement for TolB-CL interactions can be partially bypassed in cells lacking CL, suggesting compensatory effects through other lipids only when CL is absent. Our findings reveal a previously unappreciated lipid-binding role for TolB, and provide novel insights into how the Tol-Pal complex may facilitate phospholipid transport across the cell envelope. Our work will inform future strategies towards developing new antibiotics against Gram-negative bacteria.

Guanosine 5-monophosphate (GMP) synthetases, enzymes that catalyze the conversion of xanthosine 5-monophosphate (XMP) to GMP are comprised of two different catalytic units, which are either two domains of a polypeptide chain or two subunits that associate to form a complex. The glutamine amidotransferase (GATase) unit hydrolyzes glutamine generating ammonia and the ATP pyrophosphatase (ATPPase) unit catalyzes the formation of AMP-XMP intermediate. The substrate-bound ATPPase allosterically activates GATase and the ammonia thus generated is tunnelled to the ATPPase active site where it reacts with AMP-XMP generating GMP. In ammonia tunnelling enzymes reported thus far, a tight complex of the two subunits is observed, while the interaction of the two subunits of Methanocaldococcus jannaschii GMP synthetase (MjGMPS) is transient with the underlying mechanism of allostery and substrate channelling largely unclear. Here, we present a mechanistic model encompassing the various steps in the catalytic cycle of MjGMPS based on biochemical experiments, crystal structure and cross-linking mass spectrometry guided integrative modelling. pH dependence of enzyme kinetics establish that ammonia is tunnelled across the subunits with the lifetime of the complex being [≤] 0.5 s. The crystal structure of XMP-bound ATPPase subunit reported herein highlights the role of conformationally dynamic loops in enabling catalysis. The structure of MjGMPS derived using restraints obtained from cross-linking mass spectrometry has enabled the visualization of subunit interactions that enable allostery under catalytic conditions. We integrate the results and propose a functional mechanism for MjGMPS detailing the various steps involved in catalysis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/481963v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1eb7261org.highwire.dtl.DTLVardef@a25d02org.highwire.dtl.DTLVardef@1885ed7org.highwire.dtl.DTLVardef@ab189_HPS_FORMAT_FIGEXP M_FIG C_FIG

Computational protein design is growing in popularity as a means to engineer enzymes. Currently, protein design algorithms can predict the stability and function of the enzymes to only a limited degree. Thus, further experimental data is required for training software to more accurately characterize the structure-function relationship of enzymes. To date, the Design2Data (D2D) database holds 129 single point mutations of {beta}-glucosidase B (BglB) characterized by kinetic and thermal stability biophysical parameters. In this study, we introduced six mutants into the BglB database and examined their catalytic activity and thermal stability: L171M, H178M, M221L, E406W, N160E, and F415M.

This study explores computational design predictions related to experimental enzyme behavior by analyzing seven single-point mutants of {beta}-glucosidase B (BglB) from Paenibacillus polymyxa: Y333F, A88E, L219Q, A408H, Y173L, E340S, and Y422F. Each mutation was modeled using Foldit Standalone, and mutant selections were based on predicted thermodynamic stability changes of interest. Six of the seven mutants in this set yielded soluble, expressed protein. Most variants had similar catalytic efficiency compared to the wild type with one exception. The melting temperatures for most variants were also similar to the wild type. Correlation analysis revealed weak but potentially informative relationships between predicted {Delta}TSE and (a) thermal stability and (b) catalytic efficiency. These results further support known limitations of TSE score as a tool for single point mutation design and add to a growing dataset being generated to build the next generation of functionally predictive protein models.

A key goal of protein engineering is to accurately model the stability and catalytic activity of enzymes. However, the limitations of functional predictive abilities pose a major challenge for modeling algorithm design, and can be attributed to the lack of large data sets quantifying the functional properties of enzymes. Here, the thermal stability (TM) and Michaelis-Menten constants (kcat, KM, and kcat/KM) of six new variants of the {beta}-glucosidase B (BglB) protein are quantitatively characterized. Molecular stability of the enzyme variants were hypothesized using the Foldit software and BglB was synthesized in E. coli cells. Testing was done through a colorimetric kinetic assay and thermal stability fluorescence-based protein unfolding assay. Results from the assays suggest that all mutations, with the exception of variant Y169M, all experienced reduced catalytic efficiency compared to the wildtype. Assay results indicate that variant W123R is more thermally stable compared to the wildtype, while the differences in thermal stability between the other variants, and the wildtype are negligible. The collected thermal stability and catalytic efficiency data has been added to a data set with the aim of improving Rosetta algorithms for modeling and predicting the functional interactions between biomolecules through a machine learning algorithm and facilitate the precise engineering of protein catalysts.

Overexpression of the multidrug efflux pump MtrCDE, a critical factor of multidrug-resistance in Neisseria gonorrhoeae, the causative agent of gonorrheae, is repressed by the transcriptional regulator, MtrR (multiple transferable resistance repressor). Here, we report the results from a series of in vitro experiments to identify innate, human inducers of MtrR and to understand the biochemical and structural mechanisms of the gene regulatory function of MtrR. Isothermal titration calorimetry experiments reveal that MtrR binds the hormonal steroids progesterone, {beta}-estradiol, and testosterone, all of which are present at significant concentrations at urogenital infection sites as well as ethinyl estrogen, a component of some birth control pills. Binding of these steroids results in decreased affinity of MtrR for cognate DNA, as demonstrated by fluorescence polarization-based assays. The crystal structures of MtrR bound to each steroid provided insight into the flexibility of the binding pocket, elucidated specific residue-ligand interactions, and revealed the conformational consequences of the induction mechanism of MtrR. Three residues, D171, W136 and R176 are key to the specific binding of these gonadal steroids. These studies provide a molecular understanding of the transcriptional regulation by MtrR that promotes N. gonorrhoeae survival in its human host.

Though ribosomes have several features that help them maintain their reading frame, these safeguards can be bypassed by RNA structures that promote -1 programmed ribosomal frameshifting (-1PRF). We recently found that conformational transitions in the nascent polypeptide can enhance -1PRF, though its unclear whether this feedback plays a general role in translational recoding. Here we demonstrate that the translocation of nascent transmembrane domains is sufficient to induce -1PRF during the decoding of slippery heptamers. We identify thousands of motifs that potentially trigger -1PRF along with proteomic identifications of 33 predicted human frameshift products. We also identify thousands of splicing-dependent motifs and demonstrate that the splicing-mediated reconfiguration of transmembrane domains alters -1PRF. Finally, we show that most transcripts bearing these motifs are sensitive to the nonsense-mediated decay regulator UPF1, suggesting they modulate mRNA turnover. Our findings show that the misassembly of growing polypeptides can trigger -1PRF, premature termination, and transcript decay.

Polyketide synthases (PKSs) are megaenzymes that form chemically diverse polyketides and are found within the genomes of nearly all classes of life. We recently discovered the type I PKS from the apicomplexan parasite Toxoplasma gondii, TgPKS2, which contains a unique putative chain release mechanism that includes ketosynthase (KS) and thioester reductase (TR) domains. Our bioinformatic analysis of the thioester reductase of TgPKS2, TgTR, suggests differences in putative apicomplexan reductase domains compared to other systems and hints at a possibly conserved release mechanism within the apicomplexan subclass Coccidia. To evaluate this release module, we first isolated TgTR and observed that it is capable of 4 electron (4e-) reduction of octanoyl-CoA to the primary alcohol, octanol, utilizing NADH as a cofactor. TgTR was also capable of generating octanol in the presence of octanal and NADH, but no reactions were observed when NADPH was supplied as a cofactor. To biochemically characterize the protein, we measured the catalytic efficiency of TgTR using a fluorescence assay and determined the TgTR binding affinity for cofactor and substrates using isothermal titration calorimetry (ITC). We additionally show that TgTR is capable of reducing an acyl carrier protein (ACP)-tethered substrate by liquid chromatography mass spectrometry and determine that TgTR binds to holo-TgACP4, its predicted cognate ACP, with a KD of 5.75 {+/-} 0.77 {micro}M. Finally, our transcriptional analysis shows that TgPKS2 is upregulated [~]4-fold in the parasites cyst-forming bradyzoite stage compared to tachyzoites. Our study identifies features that distinguish TgPKS2 from well-characterized systems in bacteria and fungi, and suggests it aids the T. gondii cyst stage. Together, this work increases our knowledge of PKS thioester reductase domains and advances our understanding of unconventional polyketide chain termination mechanisms.

Multiple paralogous ABCF ATPases are encoded in most genomes, but the physiological functions remain unknown for most of them. We herein compare the four Escherichia coli K12 ABCFs - EttA, Uup, YbiT, and YheS - using assays previously employed to demonstrate EttA gates the first step of polypeptide elongation on the ribosome dependent on ATP/ADP ratio. A {Delta}uup knockout, like {Delta}ettA, exhibits strongly reduced fitness when growth is restarted from long-term stationary phase, but neither {Delta}ybiT nor {Delta}yheS exhibits this phenotype. All four proteins nonetheless functionally interact with ribosomes based on in vitro translation and single-molecule fluorescence resonance energy transfer experiments employing variants harboring glutamate-to-glutamine active-site mutations (EQ2) that trap them in the ATP-bound conformation. These variants all strongly stabilize the same global conformational state of a ribosomal elongation complex harboring deacylated tRNAVal in the P site. However, EQ2-Uup uniquely exchanges on/off the ribosome on a second timescale, while EQ2-YheS-bound ribosomes uniquely sample alternative global conformations. At sub-micromolar concentrations, EQ2-EttA and EQ2-YbiT fully inhibit in vitro translation of an mRNA encoding luciferase, while EQ2-Uup and EQ2-YheS only partially inhibit it at ~10-fold higher concentrations. Moreover, tripeptide synthesis reactions are not inhibited by EQ2-Uup or EQ2-YheS, while EQ2-YbiT inhibits synthesis of both peptide bonds and EQ2-EttA specifically traps ribosomes after synthesis of the first peptide bond. These results support the four E. coli ABCF paralogs all having different activities on translating ribosomes, and they suggest that there remains a substantial amount of functionally uncharacterized "dark matter" involved in mRNA translation.

Penicillin-binding proteins (PBPs) are an essential family of bacterial enzymes that are inhibited by the {beta}-lactam class of antibiotics. PBP inhibition disrupts cell wall biosynthesis, which results in deficient growth and proliferation, and ultimately leads to lysis. IC50 values are often employed as descriptors of enzyme inhibition and inhibitor selectivity but can be misleading in the study of time-dependent, irreversible inhibitors. Due to this disconnect, the second order rate constant kinact/KI is a more appropriate metric of covalent inhibitor potency. Despite being the gold standard measurement of potency, kinact/KI values are typically obtained from in vitro assays, which limits assay throughput if investigating an enzyme family with multiple homologs (such as the PBPs). Therefore, we developed a whole-cell kinact/KI assay to define inhibitor potency for the PBPs in Streptococcus pneumoniae using the fluorescent activity-based probe Bocillin-FL. Our results align with in vitro kinact/KI data and show a comparable relationship to previously established IC50 values. These results support the validity of our in vivo kinact/KI method as a means of obtaining a full picture of {beta}-lactam potency for a suite of PBPs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/592586v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@e265ecorg.highwire.dtl.DTLVardef@1917decorg.highwire.dtl.DTLVardef@e2253forg.highwire.dtl.DTLVardef@fb6c60_HPS_FORMAT_FIGEXP M_FIG C_FIG

The last step in the biosynthesis of the lipoyl cofactor (LipCo) is the addition of two sulfur atoms at C6 and C8 of an n-octanoyl chain attached in an amide linkage to a target lysyl residue of a lipoyl carrier protein. The enzyme that catalyzes this reaction, lipoyl synthase (LipA in bacteria, and LIAS in humans), is a member of the radical S-adenosylmethionine (SAM) superfamily. As such, it requires a [4Fe-4S] cluster cofactor to cleave SAM reductively to generate two 5'-deoxyadenosyl 5'-radicals (5'-dA*) that abstract the C6 and C8 hydrogen atoms (H*) of the substrate in two distinct steps. LipAs also contain a second [4Fe-4S] cluster, termed the auxiliary cluster, degraded during turnover as the source of the attached sulfur atoms. The auxiliary cluster is ligated by three cysteines in a CX4CX5C motif and one serine residue (Ser308 in Escherichia coli) in a highly conserved R306SS308Y motif in the C-terminal region of the protein. Here, we show that Arg306 and Ser308 are absolutely required for LipCo formation. Substitution of Arg306 with Lys results in a protein that is essentially inactive due to poor substrate binding and positioning in the active site. Multiple different substitutions of Ser308 were engineered. Most notable were the S308C and S308A variants, which gave greatly diminished LipCo formation. However, the S308C variant resulted in greater production of the 6-mercaptooctanoyl peptide, an intermediate in the reaction, and the formation of a desaturated product, determined to be a 6-octenoyl group attached to the peptide substrate. Furthermore, the 3Fe cluster formed during cannibalization of the auxiliary cluster during C6 sulfur substitution in the wild-type reaction is not observed with the S308C variant. Instead, the auxiliary cluster remains tetranuclear and forms a monothiolated cross-linked species with a high-spin, S = 7/2, configuration that decays to the 6-octenoyl-containing product. Other amino acids in the RSSY motif were not essential for catalysis.

One of the most essential biomolecular functions is the ribosome translocation for protein synthesis, in which the ribosome moves on the mRNA by primarily three nucleotides per step in the presence of elongation factor G (EF-G). The large conformational changes of EF-G generate significant mechanical force, referred to as power stroke. Quantification of power stroke remains under debate and its correlation with translocation fidelity has not been observed. In this work, we use quantum sensing techniques for measuring both the EF-G power stroke and its influence on ribosome translocation steps. Two EF-G mutants, H584K and Q508K, were expressed, with the mutated residues directly interacting with tRNA. H584K, which interacts on codon-anticodon minihelix, produced less power stroke of 60{+/-}6 pN and induced "-1" frameshifting, wherein the ribosome translocated only two nucleotides. In contrast, Q508K, which interacts with tRNA residue 37 immediately outside the codon-anticodon minihelix, exhibited a normal power stroke of 89{+/-}11 pN and maintained canonical 3-nucleotide translocation. These findings provide direct mechanistic evidence that the pivotal point and EF-G power stroke are critical for maintaining translocation fidelity and highlight the potential of quantum sensing for chemical biology.

The relatively small size and scope of most current datasets of biophysical mutation effects in enzymes limit the ability to develop data-driven algorithms enabling accurate generative modeling tools for designing novel enzyme function. Here, the Michaelis-Menten constants (kcat, KM, and kcat/KM) and thermal stability (TM) of five new mutations of {beta}-glucosidase B from Paenibacillus polymyxa (BglB) are characterized. Foldit software was used to create molecular models of the mutants, for which synthetic genes were constructed and the corresponding proteins produced and purified from E. coli. It was found that mutations that disrupted pre-existing hydrogen bonds near the active site had reduced expression in contrast to mutations at the same site that did not affect native hydrogen bonding. This is consistent with previous results showing the relationship between hydrogen bonding and enzyme functionality. These mutants contribute to a growing data set of >100 mutants that have been characterized for expression, kinetic, and thermal properties