Chemosensory protein 22 in Riptortus pedestris is involved in the recognition of three soybean volatiles.

Riptortus pedestris (Hemiptera: Alydidae), a common agricultural pest, is the major causative agent of "soybean staygreen." However, the interactions between chemosensory proteins (CSPs) in R. pedestris and host plant volatiles have yet to be comprehensively studied. In this study, we performed real-time fluorescence quantitative polymerase chain reaction (PCR) to analyze the antennal expression of RpedCSP22 and subsequently analyzed the interactions between 21 soybean volatiles, five aggregation pheromones, and RpedCSP22 protein in vitro using a protein expression system, molecular docking, site-directed mutagenesis, and fluorescence competitive binding experiments. The RpedCSP22 protein showed binding affinity to three soybean volatiles (benzaldehyde, 4-ethylbenzaldehyde, and 1-octene-3-ol), with optimal binding observed under neutral pH conditions, and lost binding ability after site-directed mutagenesis. In subsequent RNA interference (RNAi) studies, gene silencing was more than 90 %, and in silenced insects, electroantennographic responses were reduced by more than 75 % compared to non-silenced insects. Moreover, Y-tube olfactory behavioral assessments revealed that the attraction of R. pedestris to the three soybean volatiles was significantly attenuated. These findings suggest that RpedCSP22 plays an important role in the recognition of host plant volatiles by R. pedestris andprovides a theoretical basis for the development of novel inhibitors targeting pest behavior.

Efficient cloning of linear DNA inserts (ECOLI) into plasmids using site-directed mutagenesis.

This study introduces a novel cost-effective technique for cloning of linear DNA plasmid inserts, aiming to address the associated expenses linked with popular in vitro DNA assembly methods. Specifically, we introduce ECOLI (Efficient Cloning Of Linear Inserts), a method utilizing a PCR product-based site-directed mutagenesis. In comparison to other established in vitro DNA assembly methods, our approach is without the need for costly synthesis or specialized kits for recombination or restriction sites. ECOLI offers a fast, efficient, and economical alternative for cloning inserts up to several hundred nucleotides into plasmid constructs, thus enhancing cloning accessibility and efficiency. This method can enhance molecular biology research, as we briefly demonstrated on the Dishevelled gene from the WNT signaling pathway.

[Site-directed mutagenesis enhances the activity of dextranase from Arthrobacter oxidans KQ11].

Dextranase is an enzyme that specifically hydrolyzes the α-1, 6 glucoside bond. In order to improve the activity of dextranase from Arthrobacter oxidans KQ11, site-directed mutagenesis was used to modify the amino acids involved in the "tunnel-like binding site". A saturating mutation at position 507 was carried out on this basis. The mutant enzymes A356G, S357W, W507Y, and W507F with improved enzyme activities and catalytic efficiency were successfully obtained. Compared with wild type (WT), W507Y showed the specific activity increasing by 3.00 times, the kcat value increasing by 3.62 times, the Km value decreasing by 54%, and the catalytic efficiency (kcat/Km) increasing by 8.98 times. The three-dimensional structure analysis showed that the increase of the number of hydrogen bonds and the distance between "tunnel-like binding sites" were important factors affecting enzyme activity. Compared with WT, W507Y had a shortened distance from the residues on the other side of the "tunnel-like binding site", which made it easier to generate hydrogen binding forces. Accordingly, the substrate hydrolysis and product efflux were accelerated, which dramatically increased the enzyme activity and catalytic efficiency.

Two types of amino acid substitutions in the succinate dehydrogenase complex subunit confer resistance to benzovindiflupyr in Colletotrichum sublineola.

BACKGROUND: Colletotrichum sublineola is the pathogenic fungus that causes sorghum anthracnose, which seriously threatens sorghum yield. Benzovindiflupyr is a succinate dehydrogenase inhibitor with good control effects on various crop diseases. However, the control of sorghum anthracnose by benzovindiflupyr and the risk of resistance to benzovindiflupyr in this pathogen are not well studied. Therefore, this study aimed to evaluate the benzovindiflupyr resistance and underlying mechanisms in C. sublineola. RESULTS: Analysis of the sensitivity of 126 C. sublineola strains to benzovindiflupyr revealed that the average EC50 of the fungicide was 0.0503 ± 0.0189 µg mL-1, with a unimodal normal distribution curve. The survival fitness of 10 benzovindiflupyr-resistant strains decreased to varying degrees compared with that of the wild-type parental strains. Additionally, a significant positive cross-resistance was observed between benzovindiflupyr and carboxin. Sequencing analyses identified two mutation sites, CsSdhBH249Y and CsSdhCG81V, in the resistant strains. Further molecular docking and site-directed mutagenesis experiments confirmed that the CsSdhBH249Y and CsSdhCG81V substitutions conferred resistance to benzovindiflupyr in C. sublineola. CONCLUSION: Colletotrichum sublineola is sensitive to benzovindiflupyr and shows a moderate resistance risk to benzovindiflupyr. Two specific point substitutions, CsSdhBH249Y and CsSdhCG81V, are responsible for the resistance of C. sublineola to benzovindiflupyr. These findings offer a theoretical foundation for strategic application of the fungicide in controlling sorghum anthracnose, and for potentially delaying the emergence and progression of resistance. © 2024 Society of Chemical Industry.

Quinone chemistry in respiratory complex I involves protonation of a conserved aspartic acid residue.

Respiratory complex I is a central metabolic enzyme coupling NADH oxidation and quinone reduction with proton translocation. Despite the knowledge of the structure of the complex, the coupling of both processes is not entirely understood. Here, we use a combination of site-directed mutagenesis, biochemical assays, and redox-induced FTIR spectroscopy to demonstrate that the quinone chemistry includes the protonation and deprotonation of a specific, conserved aspartic acid residue in the quinone binding site (D325 on subunit NuoCD in Escherichia coli). Our experimental data support a proposal derived from theoretical considerations that deprotonation of this residue is involved in triggering proton translocation in respiratory complex I.

Identification of human-specific amino acid residues governing atenolol transport via organic cation transporter 2.

Organic cation transporter 2 (OCT2/SLC22A2) is predominantly localized on the basolateral membranes of renal tubular epithelial cells and plays a crucial role in the renal secretion of various cationic drugs. Although variations in substrate selectivity among renal organic cation transport systems across species have been reported, the characteristics of OCT2 remain unclear. In this study, we demonstrated that atenolol, a ß1-selective adrenergic antagonist, is transported almost exclusively by human OCT2, contrasting with OCT2s from other selected species. Using chimeric constructs between human OCT2 (hOCT2) and the highly homologous monkey OCT2 (monOCT2), along with site-directed mutagenesis, we identified non-conserved amino acids Val8, Ala31, Ala34, Tyr222, Tyr245, Ala270, Ile394, and Leu503 as pivotal for hOCT2-mediated atenolol transport. Kinetic analysis revealed that atenolol was transported by hOCT2 with a 12-fold lower affinity than MPP+, a typical OCT2 substrate. The inhibitory effect of atenolol on MPP+ transport was 6200-fold lower than that observed for MPP+ on atenolol transport. Additionally, we observed weaker inhibitory effects on MPP+ transport compared to atenolol transport with ten different OCT2 substrates. Altogether, this study suggests that eight hOCT2-specific amino acids constitute the low-affinity recognition site for atenolol transport, indicating differences in OCT2-mediated drug elimination between humans and highly homologous monkeys. Our findings underscore the importance of understanding species-specific differences in drug transport mechanisms, shedding light on potential variations in drug disposition and aiding in drug development.

Effect of Cu(II) and Conserved Copper Binding Sites on the Multicopper Oxidase CopA and Characterization of BioMnOx.

The microbial manganese removal process is believed to consist of the catalytic oxidation of Mn(II) by manganese oxidase. In this study, the multicopper oxidase CopA was purified and exhibited high manganese oxidation activity in vitro, and it was found that Cu(II) can significantly enhance its manganese oxidation activity. Gene site-directed mutagenesis was used to mutate four conserved copper binding sites of CopA to obtain four mutant strains. The manganese removal efficiencies of the four strains were determined, and it was found that H120 is the catalytically active site of CopA. The loss of Cu(II) and the mutation of the conserved copper binding site H120 resulted in the loss of ethoxyformyl and quinone modifications, a reduction in the number of modifications, and a change in the position of modifications, eventually causing a decrease in protein activity from 85.87% to 70.1%. These results reveal that Cu(II) and H120 play an indispensable role in manganese oxidation by the multicopper oxidase CopA. X-ray photoelectron spectroscopy (XPS) analysis indicates that biogenic manganese oxides produced by strains and by CopA were both composed of MnO2 and Mn3O4 and that the average valence of Mn was 3.2.

Construction of highly active and stable recombinant nattokinase by engineered bacteria and computational design.

Nattokinase (NK) is an enzyme that has been recognized as a new potential thrombolytic drug due to its strong thrombolytic activity. However, it is difficult to maintain the enzyme activity of NK during high temperature environment of industrial production. In this study, we constructed six NK mutants with potential for higher thermostability using a rational protein engineering strategy integrating free energy-based methods and molecular dynamics (MD) simulation. Then, wild-type NK and NK mutants were expressed in Escherichia coli (E. coli), and their thermostability and thrombolytic activity were tested. The results showed that, compared with wild-type NK, the mutants Y256P, Q206L and E156F all had improved thermostability. The optimal mutant Y256P showed a higher melting temperature (Tm) of 77.4 °C, an increase of 4 °C in maximum heat-resistant temperature and an increase of 51.8 % in activity at 37 °C compared with wild-type NK. Moreover, we also explored the mechanism of the increased thermostability of these mutants by analysing the MD trajectories under different simulation temperatures.

Circular PCR as an efficient and precise umbrella of methods for the generation of circular dsDNA with staggered nicks: Mechanism and types.

Here, we introduce the highly versatile circular polymerase chain reaction (CiPCR) technique, propose a mechanism of action, and describe a number of examples demonstrating the versatility of this technique. CiPCR takes place between two fragments of dsDNA with two homologous regions, as long as one of the fragments carries said regions at its 3'- and 5'-ends. Upon hybridization, elongation by a polymerase occurs from all 3'-ends continuously until a 5'-end is reached, leading to stable circular dsDNA with staggered nicks. When both dsDNA fragments carry the homology at their 3'- and 5'-ends (Type I CiPCR), all four 3'-ends effectively prime amplification of the intervening region and CiPCR products can function as template during the reaction. In contrast, when only one of the two dsDNA fragments carries the homologous regions at its 3'- and 5'-ends and the other carries such regions internally (Type II CiPCR), only two 3'-ends can be amplified and CiPCR products possess no template activity. We demonstrate the applicability of both CiPCR types via well-illustrated experimental examples. CiPCR is well adapted to the quick resolution of most of the molecular cloning challenges faced by the biology/biomedicine laboratory, including the generation of insertions, deletions, and mutations.

Expression Pattern of RpCSP6 from Rhopalosiphum padi and Its Binding Mechanism with Deltamethrin: Insights into Chemosensory Protein-Mediated Insecticide Resistance.

The mechanisms of insecticide resistance are complex. Recent studies have revealed a novel mechanism involving the chemosensory system in insecticide resistance. However, the specific binding mechanism between olfactory-related genes and insecticides needs to be clarified. In this study, the binding mechanism between pyrethroid insecticide deltamethrin and RpCSP6 from Rhopalosiphum padi was investigated by using computational and multiple experimental methods. RpCSP6 was expressed in different tissues and developmental stages of R. padi and can be induced by deltamethrin. Knockdown of RpCSP6 significantly increased the susceptibility of R. padi to deltamethrin. The binding affinity of RpCSP6 to 24 commonly used insecticides was measured. Seven key residues were found to steadily interact with deltamethrin, indicating their significance in the binding affinity to the insecticide. Our research provided insights for effectively analyzing the binding mechanism of insect CSPs with insecticides, facilitating the development of new and effective insecticides that target insect CSPs.

Structural and functional analysis of plant ELO-like elongase for fatty acid elongation.

ELO-like elongase is a condensing enzyme elongating long chain fatty acids in eukaryotes. Eranthis hyemalis ELO-like elongase (EhELO1) is the first higher plant ELO-type elongase that is highly active in elongating a wide range of polyunsaturated fatty acids (PUFAs) and some monounsaturated fatty acids (MUFAs). This study attempted using domain swapping and site-directed mutagenesis of EhELO1 and EhELO2, a close homologue of EhELO1 but with no apparent elongase activity, to elucidate the structural determinants critical for catalytic activity and substrate specificity. Domain swapping analysis of the two showed that subdomain B in the C-terminal half of EhELO1 is essential for MUFA elongation while subdomain C in the C-terminal half of EhELO1 is essential for both PUFA and MUFA elongations, implying these regions are critical in defining the architecture of the substrate tunnel for substrate specificity. Site-directed mutagenesis showed that the glycine at position 220 in the subdomain C plays a key role in differentiating the function of the two elongases. In addition, valine at 161 and cysteine at 165 in subdomain A also play critical roles in defining the architecture of the deep substrate tunnel, thereby contributing significantly to the acceptance of, and interaction with primer substrates.

Mutagenesis on a complex mouse genetic background by site-specific nucleases.

Mouse models with complex genetic backgrounds are increasingly used in preclinical research to accurately model human disease and to enable temporal and cell-specific evaluation of genetic manipulations. Backcrossing mice onto these complex genetic backgrounds takes time and leads to significant wastage of animals. In this study, we aimed to evaluate whether site-specific nucleases could be used to generate additional genetic mutations in a complex genetic background, using the REVERSA mouse model of atherosclerosis, a model harbouring four genetically altered alleles. The model is comprised of a functional null mutation in the Ldlr gene in combination with a ApoB100 allele, which, after high-fat diet, leads to the rapid development of atherosclerosis. The regression of the pathology is achieved by inducible knock-out of the Mttp gene. Here we report an investigation to establish if microinjection of site-specific nucleases directly into zygotes prepared from the REVERSA could be used to investigate the role of the ATP binding cassette transporter G1 (ABCG1) in atherosclerosis regression. We show that using this approach we could successfully generate two independent knockout lines on the REVERSA background, both of which exhibited the expected phenotype of a significant reduction in cholesterol efflux to HDL in bone marrow-derived macrophages. However, loss of Abcg1 did not impact atherosclerosis regression in either the aortic root or in aortic arch, demonstrating no important role for this transporter subtype. We have demonstrated that site-specific nucleases can be used to create genetic modifications directly onto complex disease backgrounds and can be used to explore gene function without the need for laborious backcrossing of independent strains, conveying a significant 3Rs advantage.

Characterization of urease active calcite-producing strain YX-3 combined with the whole genome.

Urease found in a wide range of microorganisms plays a vital role in ureolytic induced calcite precipitation (UICP). However, the genomic information on urease-producing strains is limited, and there is a need for further in-depth studies on aspects such as the regulation of urease activity by nickel ligand residues. The present study delved into the elucidation of urease activity in a newly isolated strain YX-3 coupled with nickel-ligand residues by employing the genetic architecture of biomineralization-controlled growth, molecular docking, molecular dynamics simulation (MDS), and site-directed mutagenesis. Genome-wide sequencing showed the presence of urease gene clusters, comprising structural genes ureA, ureB, and ureC, alongside auxiliary genes ureD, ureE, ureF, and ureG. RT-qPCR analysis showed that the addition of NiCl2 resulted in a significant up-regulation of ureC expression. His267, His294, and Gly325 in the domain of UreC were further proved to coordinate with nickel ions and urea simultaneously through homology modeling and molecular docking, and molecular dynamics simulations (MDS) showed the urease-urea docking complexes exhibited degressive binding stability by four metrics including root mean square deviations (RMSD) when those residues were mutated into alanine respectively. Western blotting exhibited that mutations of H267A, H294A, and G325A led to a reduction in the relative expression of urease, wherein urease activity was about 62%, 45%, and 20% times that of the wild type (WT), respectively. The overexpression results further confirmed the importance of these residues for urease activity and CaCO3 precipitation. These results would help to deepen the understanding of urease-producing strains at a molecular level and expand the theoretical basis for modulating urease activity.

Engineering of Recombinant Human Papillomavirus 16 L1 Protein for Incorporation with para-Azido-L-Phenylalanine.

Human papillomavirus (HPV) L1 capsid protein were produced in several host systems, but few studies have focused on enhancing the properties of the L1 protein. In this study, we aimed to produce recombinant Human papillomavirus (HPV) L1 capsid protein containing para-azido-L-phenylalanine (pAzF) in Escherichia coli. First, we expressed the maltose-binding protein (MBP)- fused HPV16 L1, and 5 residues in HPV16 L1 protein were selected by the in silico modeling for amber codon substitution. Among the variants of the five locations, we identified a candidate that exhibited significant differences in expression with and without pAzF via genetic code expansion (GCE). The expressed recombinant MBP-HPV16L1 protein was confirmed for incorporation of pAzF and the formation of VLPs was tested in vitro.

Molecular mechanisms for the selective transport of dichlorofluorescein by human organic anion transporting polypeptide 1B1.

Human organic anion transporting polypeptide 1B1 (OATP1B1) and 1B3 are two highly homologous liver-specific uptake transporters. However, 2',7'-dichlorofluorescein (DCF) is preferably transported by OATP1B1. In the current study, the molecular mechanisms for the selective transport of DCF by OATP1B1 were investigated by constructing and characterizing an array of OATP1B1/1B3 chimeras and site-directed mutagenesis. Our results show that transmembrane domain 10 (TM10) is crucial for the surface expression and function of OATP1B1, in which Q541 and L545 play the most important roles in DCF transport. Replacement of TM10 in OATP1B1 with its OATP1B3 counterpart led to OATP1B1's complete intracellular retention. Q541 and L545 may interact with DCF directly via hydrogen bonding and hydrophobic interactions. The decrease of DCF uptake by Q541A and L545S was due to their reduced binding affinity for DCF as compared to OATP1B1. In addition, Q541 and L545 are also crucial for the transport of estradiol-17ß-glucuronide (E17ßG) but not for the transport of estrone-3-sulfate (E3S), indicating different interaction modes between DCF/E17ßG and E3S in OATP1B1. Taken together, Q541 and L545 in TM10 are critical for OATP1B1-mediated DCF uptake, but their effect is substrate-dependent. Significance Statement The key transmembrane domains (TMs) and amino acid residues for the selective transport of DCF by OATP1B1 were identified. TM10 is crucial for the surface expression and function of OATP1B1. Within TM10, Q541 and L545 played the most significant roles and affected the function of OATP1B1 in a substrate-dependent manner. This information is crucial for a better understanding of the mechanism of the multispecificity of OATP1B1 and as a consequence the mechanism of OATP1B1-mediated drug-drug interactions.

Understanding the role of negative charge in the scaffold of an artificial enzyme for CO2 hydrogenation on catalysis.

We have approached the construction of an artificial enzyme by employing a robust protein scaffold, lactococcal multidrug resistance regulator, LmrR, providing a structured secondary and outer coordination spheres around a molecular rhodium complex, [RhI(PEt2NglyPEt2)2]-. Previously, we demonstrated a 2-3 fold increase in activity for one Rh-LmrR construct by introducing positive charge in the secondary coordination sphere. In this study, a series of variants was made through site-directed mutagenesis where the negative charge is located in the secondary sphere or outer coordination sphere, with additional variants made with increasingly negative charge in the outer coordination sphere while keeping a positive charge in the secondary sphere. Placing a negative charge in the secondary or outer coordination sphere demonstrates decreased activity by a factor of two compared to the wild-type Rh-LmrR. Interestingly, addition of positive charge in the secondary sphere, with the negatively charged outer coordination sphere restores activity. Vibrational and NMR spectroscopy suggest minimal changes to the electronic density at the rhodium center, regardless of inclusion of a negative or positive charge in the secondary sphere, suggesting another mechanism is impacting catalytic activity, explored in the discussion.

Engineering of the Phytase YiAPPA to Improve Thermostability and Activity and Its Application Potential in Dephytinization of Food Ingredients.

The aim of this study was to modify phytase YiAPPA via protein surficial residue mutation to obtain phytase mutants with improved thermostability and activity, enhancing its application potential in the food industry. First, homology modeling of YiAPPA was performed. By adopting the strategy of protein surficial residue mutation, the lysine (Lys) and glycine (Gly) residues on the protein surface were selected for site-directed mutagenesis to construct single-site mutants. Thermostability screening was performed to obtain mutants (K189R and K216R) with significantly elevated thermostability. The combined mutant K189R/K216R was constructed via beneficial mutation site stacking and characterized. Compared with those of YiAPPA, the half-life of K189R/K216R at 80°C was extended from 14.81 min to 23.35 min, half-inactivation temperature (T50 30) was increased from 55.12°C to 62.44°C, and Tm value was increased from 48.36°C to 53.18°C. Meanwhile, the specific activity of K189R/K216R at 37°C and pH 4.5 increased from 3960.81 to 4469.13 U/mg. Molecular structure modeling analysis and molecular dynamics simulation showed that new hydrogen bonds were introduced into K189R/K216R, improving the stability of certain structural units of the phytase and its thermostability. The enhanced activity was primarily attributed to reduced enzyme-substrate binding energy and shorter nucleophilic attack distance between the catalytic residue His28 and the phytate substrate. Additionally, the K189R/K216R mutant increased the hydrolysis efficiency of phytate in food ingredients by 1.73-2.36 times. This study established an effective method for the molecular modification of phytase thermostability and activity, providing the food industry with an efficient phytase for hydrolyzing phytate in food ingredients.

Amino acid mutation of succinate dehydrogenase complex induced resistance to benzovindiflupyr in Magnaporthe oryzae.

Magnaporthe oryzae is a rice blast pathogen that seriously threatens rice yield. Benzovindiflupyr is a succinate dehydrogenase inhibitor (SDHI) fungicide that effectively controls many crop diseases. Benzovindiflupyr has a strong inhibitory effect on M. oryzae; however, control of rice blast by benzovindiflupyr and risk of resistance to benzovindiflupyr are not well studied in this pathogen. In this study, six benzovindiflupyr-resistant strains were obtained by domestication induced in the laboratory. The MoSdhBH245D mutation was the cause of M. oryzae resistance to benzovindiflupyr, which was verified through succinate dehydrogenase (SDH) activity assays, molecular docking, and site-specific mutations. Survival fitness analysis showed no significant difference between the benzovindiflupyr-resistant and parent strains. Positive cross-resistance to benzovindiflupyr and other SDHIs and negative cross-resistance to azoxystrobin were observed. Therefore, the risk of benzovindiflupyr resistance in M. oryzae might be medium to high. It should be combined with other classes of fungicides (tebuconazole and azoxystrobin) to slow the development of resistance.

A conserved phenylalanine motif among teleost fish provides insight for improving electromagnetic perception.

Magnetoreceptive biology as a field remains relatively obscure; compared with the breadth of species believed to sense magnetic fields, it remains under-studied. Here, we present grounds for the expansion of magnetoreception studies among teleosts. We begin with the electromagnetic perceptive gene (EPG) from Kryptopterus vitreolus and expand to identify 72 teleosts with homologous proteins containing a conserved three-phenylalanine (3F) motif. Phylogenetic analysis provides insight as to how EPG may have evolved over time and indicates that certain clades may have experienced a loss of function driven by different fitness pressures. One potential factor is water type with freshwater fish significantly more likely to possess the functional motif version (FFF), and saltwater fish to have the non-functional variant (FXF). It was also revealed that when the 3F motif from the homologue of Brachyhypopomus gauderio (B.g.) is inserted into EPG-EPG(B.g.)-the response (as indicated by increased intracellular calcium) is faster. This indicates that EPG has the potential to be engineered to improve upon its response and increase its utility to be used as a controller for specific outcomes.

Evidence of isochorismate channeling between the Escherichia coli enterobactin biosynthetic enzymes EntC and EntB.

Enterobactin is a high-affinity iron chelator produced and secreted by Escherichia coli and Salmonella typhimurium to scavenge scarce extracellular Fe3+ as a micronutrient. EntC and EntB are the first two enzymes in the enterobactin biosynthetic pathway. Isochorismate, produced by EntC, is a substrate for EntB isochorismatase. By using a competing isochorismate-consuming enzyme (the E. coli SEPHCHC synthase MenD), we found in a coupled assay that residual EntB isochorismatase activity decreased as a function of increasing MenD concentration. In the presence of excess MenD, EntB isochorismatase activity was observed to decrease by 84%, indicative of partial EntC-EntB channeling (16%) of isochorismate. Furthermore, addition of glycerol to the assay resulted in an increase of residual EntB isochorismatase activity to approximately 25% while in the presence of excess MenD. These experimental outcomes supported the existence of a substrate channeling surface identified in a previously reported protein-docking model of the EntC-EntB complex. Two positively charged EntB residues (K21 and R196) that were predicted to electrostatically guide negatively charged isochorismate between the EntC and EntB active sites were mutagenized to determine their effects on substrate channeling. The EntB variants K21D and R196D exhibited a near complete loss of isochorismatase activity, likely due to electrostatic repulsion of the negatively charged isochorismate substrate. Variants K21A, R196A, and K21A/R196A retained partial EntB isochorismatase activity in the absence of EntC; in the presence of EntC, isochorismatase activity in all variants increased to near wild-type levels. The MenD competition assay of the variants revealed that while K21A channeled isochorismate as efficiently as wild-type EntB (~ 15%), the variants K21A/R196A and R196A exhibited an approximately 5-fold loss in observed channeling efficiency (~3%). Taken together, these results demonstrate that partial substrate channeling occurs between EntC and EntB via a leaky electrostatic tunnel formed upon dynamic EntC-EntB complex formation and that EntB R196 plays an essential role in isochorismate channeling.