Structure and Gating Behavior of the Human Integral Membrane Protein VDAC1 in a Lipid Bilayer.

The voltage-dependent anion channel (VDAC), the most abundant protein in the outer mitochondrial membrane, is responsible for the transport of all ions and metabolites into and out of mitochondria. Larger than any of the ß-barrel structures determined to date by magic-angle spinning (MAS) NMR, but smaller than the size limit of cryo-electron microscopy (cryo-EM), VDAC1's 31 kDa size has long been a bottleneck in determining its structure in a near-native lipid bilayer environment. Using a single two-dimensional (2D) crystalline sample of human VDAC1 in lipids, we applied proton-detected fast magic-angle spinning NMR spectroscopy to determine the arrangement of ß strands. Combining these data with long-range restraints from a spin-labeled sample, chemical shift-based secondary structure prediction, and previous MAS NMR and atomic force microscopy (AFM) data, we determined the channel's structure at a 2.2 Å root-mean-square deviation (RMSD). The structure, a 19-stranded ß-barrel, with an N-terminal α-helix in the pore is in agreement with previous data in detergent, which was questioned due to the potential for the detergent to perturb the protein's functional structure. Using a quintuple mutant implementing the channel's closed state, we found that dynamics are a key element in the protein's gating behavior, as channel closure leads to the destabilization of not only the C-terminal barrel residues but also the α2 helix. We showed that cholesterol, previously shown to reduce the frequency of channel closure, stabilizes the barrel relative to the N-terminal helix. Furthermore, we observed channel closure through steric blockage by a drug shown to selectively bind to the channel, the Bcl2-antisense oligonucleotide G3139.

Rapid and highly sensitive detection of Escherichia coli O157:H7 in food with loop-mediated isothermal amplification coupled to a new bioluminescent assay.

Testing for bioluminescent pyrophosphate is a convenient method of DNA detection without complex equipments, but it is insufficiently sensitive and offers no particular time advantage over other rapid detection methods. The shortcomings of the traditional bioluminescent pyrophosphate method have been addressed by using 2-deoxyadenosine-5-(α-thio)-triphosphate (dATPαS) instead of dATP for LAMP, thus reducing the high background signal and generating a constant background value. In this study, LAMP coupled to a novel bioluminescent pyrophosphate assay was developed to detect E. coli O157:H7. The new method has a limit of detection of <10 copies/µL or 5 CFU/mL; its sensitivity is higher than that of the conventional LAMP assay. Moreover, a food-borne pathogen can be detected when a single DNA template is included in the LAMP assay, making it 100 times more sensitive than the traditional LAMP method. Three hundred food samples were tested with this assay and the accuracy of detection was verified with a culture method and MALDI Biotyper. The assay only took 90-120 min and detected <10 copies of the pathogen. This method had the advantages of rapidity, sensitivity, and simplicity, so it is very competitive for the rapid and highly sensitive detection of food-borne pathogens.

Development of an ICP-MS/MS approach for absolute quantification and determination of phosphodiester to phosphorothioate ratio in therapeutic oligonucleotides.

A new analytical method based on ICP-MS/MS is proposed for the characterization of synthetic phosphorothioate oligonucleotides. Absolute quantification of oligonucleotides is challenging, as well as the determination of phosphodiester to phosphorothioate ratio for phosphorothioate oligonucleotides. Both are considered as critical quality attributes and should be determined using robust validated methods. The method we developed was designed to be easy to apply, fast, and robust. It allows simultaneous absolute quantification of an oligonucleotide (based on the quantification of phosphorus), determination of the phosphodiester to phosphorothioate ratio (based on the quantification of phosphorus and sulfur) and optionally determination of sodium (or any other metal) as a counter ion. The performance of the method was demonstrated on O,O-diethyl thiophosphate potassium salt, a well characterized model substance that possesses similar composition to phosphorothioate oligonucleotides. Method was also tested on different synthetic phophorothioate oligonucleotides, showing excellent accuracy and precision.

The NTP binding site of the polymerase ribozyme.

A previously developed RNA polymerase ribozyme uses nucleoside triphosphates (NTPs) to extend a primer 3'-terminus, templated by an RNA template with good fidelity, forming 3'-5'-phosphordiester bonds. Indirect evidence has suggested that the ribozyme's accessory domain binds the NTP with a highly conserved purine-rich loop. To determine the NTP binding site more precisely we evolved the ribozyme for efficient use of 6-thio guanosine triphosphate (6sGTP). 6sGTP never appeared in the evolutionary history of the ribozyme, therefore it was expected that mutations would appear at the NTP binding site, adapting to more efficient binding of 6sGTP. Indeed, the evolution identified three mutations that mediate 200-fold improved incorporation kinetics for 6sGTP. A >50-fold effect resulted from mutation A156U in the purine-rich loop, identifying the NTP binding site. This mutation acted weakly cooperative with two other beneficial mutations, C113U in the P2 stem near the catalytic site, and C79U on the surface of the catalytic domain. The preference pattern of the ribozyme for different NTPs changed when position 156 was mutated, confirming a direct contact between position 156 and the NTP. The results suggest that A156 stabilizes the NTP in the active site by a hydrogen bond to the Hoogsteen face of the NTP.

Restoration of Glucose-Stimulated Cdc42-Pak1 Activation and Insulin Secretion by a Selective Epac Activator in Type 2 Diabetic Human Islets.

Glucose metabolism stimulates cell division control protein 42 homolog (Cdc42)-p21-activated kinase (Pak1) activity and initiates filamentous actin (F-actin) cytoskeleton remodeling in pancreatic ß-cells so that cytoplasmic secretory granules can translocate to the plasma membrane where insulin exocytosis occurs. Since glucose metabolism also generates cAMP in ß-cells, the cross talk of cAMP signaling with Cdc42-Pak1 activation might be of fundamental importance to glucose-stimulated insulin secretion (GSIS). Previously, the type-2 isoform of cAMP-regulated guanine nucleotide exchange factor 2 (Epac2) was established to mediate a potentiation of GSIS by cAMP-elevating agents. Here we report that nondiabetic human islets and INS-1 832/13 ß-cells treated with the selective Epac activator 8-pCPT-2'-O-Me-cAMP-AM exhibited Cdc42-Pak1 activation at 1 mmol/L glucose and that the magnitude of this effect was equivalent to that which was measured during stimulation with 20 mmol/L glucose in the absence of 8-pCPT-2'-O-Me-cAMP-AM. Conversely, the cAMP antagonist Rp-8-Br-cAMPS-pAB prevented glucose-stimulated Cdc42-Pak1 activation, thereby blocking GSIS while also increasing cellular F-actin content. Although islets from donors with type 2 diabetes had profound defects in glucose-stimulated Cdc42-Pak1 activation and insulin secretion, these defects were rescued by the Epac activator so that GSIS was restored. Collectively, these findings indicate an unexpected role for cAMP as a permissive or direct metabolic coupling factor in support of GSIS that is Epac2 and Cdc42-Pak1 regulated.

Neutron Crystallography Detects Differences in Protein Dynamics: Structure of the PKG II Cyclic Nucleotide Binding Domain in Complex with an Activator.

As one of the main receptors of a second messenger, cGMP, cGMP-dependent protein kinase (PKG) isoforms I and II regulate distinct physiological processes. The design of isoform-specific activators is thus of great biomedical importance and requires detailed structural information about PKG isoforms bound with activators, including accurate positions of hydrogen atoms and a description of the hydrogen bonding and water architecture. Here, we determined a 2.2 Å room-temperature joint X-ray/neutron (XN) structure of the human PKG II carboxyl cyclic nucleotide binding (CNB-B) domain bound with a potent PKG II activator, 8-pCPT-cGMP. The XN structure directly visualizes intermolecular interactions and reveals changes in hydrogen bonding patterns upon comparison to the X-ray structure determined at cryo-temperatures. Comparative analysis of the backbone hydrogen/deuterium exchange patterns in PKG II:8-pCPT-cGMP and previously reported PKG Iß:cGMP XN structures suggests that the ability of these agonists to activate PKG is related to how effectively they quench dynamics of the cyclic nucleotide binding pocket and the surrounding regions.

Magnetic chitosan-based adsorbent prepared via Pickering high internal phase emulsion for high-efficient removal of antibiotics.

A novel magnetic chitosan-g-poly(2-acrylamide-2-methylpropanesulfonic acid) (CTS-g-AMPS) porous adsorbent was prepared by grafting the AMPS onto the CTS in the Fe3O4 stabilized Pickering high internal phase emulsions (Pickering-HIPEs) and used for the adsorptive removal of the antibiotics tetracycline (TC) and chlorotetracycline (CTC). The results of the structure characterization showed that porous structure of the adsorbent can be tuned easily by altering amount of Fe3O4-MNPs-M and the electrostatic attraction of between SO3- and CTC, TC was the main adsorption driving force. The adsorption capacities of the adsorbent for TC and CTC can be reached to 806.60 and 876.60mg/g in a wide pH ranged from 3.0 to 11.0, respectively. And the adsorption equilibrium can be reached within 90min for TC and 50min for CTC. The magnetic porous adsorbent had good reusability, which can still attain a high adsorption capacity of 759.82 and 842.99mg/g for TC and CTC after five consecutive adsorption cycles, respectively. Therefore, the as-prepared CTS-g-AMPS magnetic adsorbent is potential to be used for adsorption removal of antibiotics from water.

Tat-Tagged and Folate-Modified N-Succinyl-chitosan (Tat-Suc-FA) Self-assembly Nanoparticle for Therapeutic Delivery OGX-011 to A549 Cells.

The objective of this study was to develop a novel type of an antisense oligonucleotide (OGX-011) loaded Tat-tagged and folate-modified N-succinyl-chitosan (Tat-Suc-FA) nanoparticles (NPs) for improving tumor targetability. In this study, Tat-Suc-FA/OGX-011NPs were prepared and its physicochemical characterizations were also evaluated. The nanoparticles showed an average diameter of 73 ± 16.6 nm, the zeta potential of +23.6 ± 0.3 mV, and a high entrapment efficiency of 89.6 ± 6.6%. Transmission electron microscopy analysis showed the nanoparticles were mostly spherical and well dispersed. The delivery efficiency of this system was investigated both in vitro and in vivo. In comparison with nontargeted Lipofectamin2000/OGX-011 and free OGX-011, Tat-Suc-FA/GOX-011 showed the highest apoptosis rate of 14.2% ± 1.8% and significant uptake in A549 cells. Tat-Suc-FA NPs loaded with GOX-011 induced significant down-regulation of s-CLU mRNA and protein levels in A549 cells. In A549 tumor-bearing mice model, Tat-Suc-FA/GOX-011 produced a more efficient down-regulation of s-CLU compared to Lipofectamin2000/OGX-011. Furthermore, the combined use of Tat-Suc-FA/OGX-011 with DDP chemotherapy showed a most significant inhibition of tumor growth and greatly enhanced the survival rate of A549 tumor-bearing mice. These findings suggested successful application of Tat-Suc-FA NPs for the high efficiency and specificity in therapeutic delivery of OGX-011 to A549 cells.

Synthesis of hydrophilic and conductive molecularly imprinted polyaniline particles for the sensitive and selective protein detection.

In this work, a novel kind of water-dispersible molecular imprinted conductive polyaniline particles was prepared through a facile and efficient macromolecular co-assembly of polyaniline with amphiphilic copolymer, and applied as the molecular recognition element to construct protein electrochemical sensor. In our strategy, an amphiphilic copolymer P(AMPS-co-St) was first synthesized using 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and styrene (St) as monomer, which could co-assemble with PANI in aqueous solution to generate PANI particles driven by the electrostatic interaction. During this process, ovalbumin (OVA) as template protein was added and trapped into the PANI NPs particles owing to their interactions, resulting in the formation of molecular imprinted polyaniline (MIP-PANI) particles. When utilizing the MIP-PANI particles as recognition element, the resultant imprinted PANI sensor not only exhibited good selectivity toward template protein (the imprinting factor α is 5.31), but also a wide linear range over OVA concentration from 10-11 to 10-6mgmL-1 with a significantly lower detection limit of 10-12mgmL-1, which outperformed most of reported OVA detecting methods. In addition, an ultrafast response time of less than 3min has also been demonstrated. The superior performance is ascribed to the water compatibility, large specific surface area of PANI particles and the electrical conductivity of PANI which provides a direct path for the conduction of electrons from the imprinting sites to the electrode surface. The outstanding sensing performance combined with its facile, quick, green preparation procedure as well as low production cost makes the MIP-PANI particles attractive in specific protein recognition and sensing.

Oxidatively-generated damage to DNA and proteins mediated by photosensitized UVA.

UVA accounts for about 95% of the solar ultraviolet (UV) radiation that reaches Earth and most likely contributes to human skin cancer risk. In contrast to UVB, which comprises the remaining 5% and is absorbed by DNA nucleobases to cause direct photodamage, UVA damages DNA indirectly. It does this largely through its interactions with cellular chromophores that act as photosensitisers to generate reactive oxygen species. Exogenously supplied chemicals, including some widely-prescribed medicines, may also act as photosensitisers and these drugs are associated with an increased risk of sun-related cancer. Because they amplify the effects of UVA on cells, they provide a means to investigate the mechanisms and effects of UVA-induced photodamage. Here, we describe some of the major lesions induced by two groups of UVA photosensitisers, the DNA thionucleotides and the fluoroquinolone antibiotics. In thionucleotides, replacement of the oxygen atoms of canonical nucleobases by sulfur converts them into strong UVA chromophores that can be incorporated into DNA. The fluoroquinolones are also UVA chromophores. They are not incorporated into DNA and induce a different range of DNA damages. We also draw attention to the potentially important contribution of photochemical protein damage to the cellular effects of photosensitised UVA. Proteins targeted for oxidation damage include DNA repair factors and we suggest that UVA-mediated protein damage may contribute to sunlight-induced cancer risk.

Crystal structure of cGMP-dependent protein kinase Iß cyclic nucleotide-binding-B domain : Rp-cGMPS complex reveals an apo-like, inactive conformation.

The R-diastereomer of phosphorothioate analogs of cGMP, Rp-cGMPS, is one of few known inhibitors of cGMP-dependent protein kinase I (PKG I); however, its mechanism of inhibition is currently not fully understood. Here, we determined the crystal structure of the PKG Iß cyclic nucleotide-binding domain (PKG Iß CNB-B), considered a 'gatekeeper' for cGMP activation, bound to Rp-cGMPS at 1.3 Å. Our structural and NMR data show that PKG Iß CNB-B bound to Rp-cGMPS displays an apo-like structure with its helical domain in an open conformation. Comparison with the cAMP-dependent protein kinase regulatory subunit (PKA RIα) showed that this conformation resembles the catalytic subunit-bound inhibited state of PKA RIα more closely than the apo or Rp-cAMPS-bound conformations. These results suggest that Rp-cGMPS inhibits PKG I by stabilizing the inactive conformation of CNB-B.

Lipid Nanoparticles Loaded with an Antisense Oligonucleotide Gapmer Against Bcl-2 for Treatment of Lung Cancer.

PURPOSE: Bcl-2 is an anti-apoptotic gene that is frequently overexpressed in human cancers. G3139 is an antisense oligonucleotide against bcl-2 that has shown limited efficacy in clinical trials. Here, we report the synthesis of a new antisense oligonucleotide containing additional chemical modifications and its delivery using nanoparticles. METHODS: An oligonucleotide G3139-GAP was synthesized, which has 2'-O-methyl nucleotides at the 5' and 3' ends based on a "gapmer" design. Furthermore, G3139-GAP was incorporated into lipid nanoparticles (LNPs) composed of DOTAP/egg PC/cholesterol/Tween 80. The LNP-loaded G3139-GAP was evaluated in A549 lung cancer cells both in vitro and in a murine xenograft model for biological activity and therapeutic efficacy. RESULTS: The LNPs showed excellent colloidal and serum stability, and high encapsulation efficiency for G3139-GAP. They have a mean particle diameter and zeta potential of 134 nm and 9.59 mV, respectively. G3139-GAP-LNPs efficiently downregulated bcl-2 expression in A549 cells, as shown by 40% and 83% reduction in mRNA and protein levels, respectively. Furthermore, G3139-GAP-LNPs were shown to inhibit tumor growth, prolong survival, and downregulate tumor bcl-2 expression in an A549 murine xenograft tumor model. These data indicate that G3139-GAP-LNPs have excellent anti-tumor efficacy and warrant further evaluation.

Thiolated alginate-based multiple layer mucoadhesive films of metformin forintra-pocket local delivery: in vitro characterization and clinical assessment.

INTRODUCTION: Periodontal disease broadly defines group of conditions in which the supportive structure of the tooth (periodontium) is destroyed. Recent studies suggested that the anti-diabetic drug metformin hydrochloride (MF) has an osteogenic effect and is beneficial for the management of periodontitis. OBJECTIVE: Development of strong mucoadhesive multiple layer film loading small dose of MF for intra-pocket application. METHODOLOGY: Multiple layer film was developed by double casting followed by compression method. Either 6% carboxy methyl cellulose sodium (CMC) or sodium alginate (ALG) constituted the inner drug (0.6%) loaded layer. Thiolated sodium alginate (TSA; 2 or 4%) constituted the outer drug free layers to enhance mucoadhesion and achieve controlled drug release. Optimized formulation was assessed clinically on 20 subjects. RESULTS: Films were uniform, thin and hard enough for easy insertion into periodontal pockets. Based on water uptake and in vitro drug release, CMC based film with 4% TSA as an outer layer was the optimized formulation with enhanced mucoadhesion and controlled drug release (83.73% over 12 h). SEM showed the effective fabrication of the triple layer film in which connective lines between the layers could be observed. FTIR examination suggests possibility of hydrogen bonding between the -NH groups of metformin and -OH groups of CMC. DSC revealed the presence of MF mainly in the amorphous form. Clinical results indicated improvement of all clinical parameters six months post treatment. CONCLUSION: The results suggested that local application of the mucoadhesive multiple layer films loaded with metformin hydrochloride was able to manage moderate chronic periodontitis.

Lipid-modified oligonucleotide conjugates: Insights into gene silencing, interaction with model membranes and cellular uptake mechanisms.

The ability of oligonucleotides to silence specific genes or inhibit the biological activity of specific proteins has generated great interest in their use as research tools and therapeutic agents. Unfortunately, their biological applications meet the limitation of their poor cellular accessibility. Developing an appropriate delivery system for oligonucleotides is essential to achieve their efficient cellular uptake. In the present work a series of phosphorothioate lipid-oligonucleotide hybrids were synthesized introducing covalently single or double lipid tails at both 3'- and 5'-termini of an antisense oligonucleotide. Gene transfections in cultured cells showed antisense luciferase inhibition without the use of a transfecting agent for conjugates modified with the double-lipid tail at 5'-termini. The effect of the double lipid-tailed modification was further studied in detail in several model membrane systems as well as in cellular uptake experiments. During these studies the spontaneous formation of self-assembled microstructures is clearly observed. Lipidation allowed the efficient incorporation of the oligonucleotide in HeLa cells by a macropinocytosis mechanism without causing cytotoxicity in cells or altering the binding properties of the oligonucleotide conjugates. In addition, both single- and double-tailed compounds showed a similar behavior in lipid model membranes, making them useful in nucleotide-based technologies.

Nucleoside 5'-O-monophosphorothioates as modulators of the P2Y14 receptor and mast cell degranulation.

Mast cells (MCs) are long-lived resident cells known for their substantial role in antigen-induced anaphylaxis and other immunoglobulin E-mediated allergic reactions as well as tumor promotion. MCs' activation results in the release of pro-inflammatory factors such as histamine, tryptase, tumor necrosis factor or carboxypeptidase A stored in secretory granules. IgE-dependent hypersensitivity has been thought to be the major pathway mediating degranulation of mast cells, but the P2Y14 nucleotide receptor activated by UDP-glucose (UDPG) may also enhance this process. In this study we identified thymidine 5'-O-monophosphorothioate (TMPS) as a molecule inhibiting UDPG-induced degranulation in a rat mast cell line (RBL-2H3). Additionally, TMPS diminished UDPG-evoked intracellular calcium mobilization in a stable HEK293T cell line overexpressing the P2Y14 receptor. Therefore, we demonstrate that the use of thymidine 5'-O-monophosphorothioate might be a novel anti-inflammatory approach based on preventingmast cell activation.

Integrated Safety Assessment of 2'-O-Methoxyethyl Chimeric Antisense Oligonucleotides in NonHuman Primates and Healthy Human Volunteers.

The common chemical and biological properties of antisense oligonucleotides provide the opportunity to identify and characterize chemical class effects across species. The chemical class that has proven to be the most versatile and best characterized is the 2'-O-methoxyethyl chimeric antisense oligonucleotides. In this report we present an integrated safety assessment of data obtained from controlled dose-ranging studies in nonhuman primates (macaques) and healthy human volunteers for 12 unique 2'-O-methoxyethyl chimeric antisense oligonucleotides. Safety was assessed by the incidence of safety signals in standardized laboratory tests for kidney and liver function, hematology, and complement activation; as well as by the mean test results as a function of dose level over time. At high doses a number of toxicities were observed in nonhuman primates. However, no class safety effects were identified in healthy human volunteers from this integrated data analysis. Effects on complement in nonhuman primates were not observed in humans. Nonhuman primates predicted safe doses in humans, but over predicted risk of complement activation and effects on platelets. Although limited to a single chemical class, comparisons from this analysis are considered valid and accurate based on the carefully controlled setting for the specified study populations and within the total exposures studied.

Proangiogenic properties of nucleoside 5'-O-phosphorothioate analogues under hyperglycaemic conditions.

Diabetes leads to impairment of the normal course of wound healing. Interestingly, recent studies have implicated a critical role of P2X/P2Y nucleotide receptors in dermal tissue regeneration and maintaining vascular homeostasis. As new vessel generation and keratinization process are decreased in diabetic patients we determined whether nucleoside 5'-O-phosphorothioate analogues might accelerate vascular endothelial growth factor (VEGF) production as well as the growth and migration of human keratinocytes under hyperglycaemic conditions. We also investigated the expression pattern of P2X/P2Y receptors in human keratinocyte HaCaT cells. We show here that nucleoside 5'-Ophosphorothioate analogues are better candidates to overcome hyperglycaemia-induced impairment of angiogenesis as compared to their unmodified counterparts. The greatest potency for VEGF release and stimulation of cell migration by thiophosphate analogues of ATP and UTP correlates with the highest P2Y2 receptor expression by HaCaT cells. We also found that UTPαS significantly increased the viability and proliferation of the HaCaT cells. These findings suggest that thiophosphate analogues of nucleotides could serve as potential therapeutic agents for promoting impaired angiogenesis under diabetic conditions.

Photocatalytic multiphase micro-droplet reactors based on complex coacervation.

We describe the synthesis and characterisation of novel photocatalytic multiphase micro-droplet reactors comprising TiO2 nanosheets dispersed in poly(diallyldimethylammonium) chloride and adenosine 5'-triphosphate or poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl complex coacervates. We demonstrate significant variations in the degree of equilibrium partitioning of small molecule dyes into the coacervate droplet systems and exploit this behaviour to successfully conduct selective photocatalytic dye degradation.

Two disparate ligand-binding sites in the human P2Y1 receptor.

In response to adenosine 5'-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7 Å resolution, and with a non-nucleotide antagonist BPTU at 2.2 Å resolution. The structures reveal two distinct ligand-binding sites, providing atomic details of P2Y1R's unique ligand-binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y1R, which is different in shape and location from the nucleotide binding site in the previously determined structure of P2Y12R, representative of another P2YR subfamily. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G-protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y1R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects.

Active site and remote contributions to catalysis in methylthioadenosine nucleosidases.

5'-Methylthioadenosine/S-adenosyl-l-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5'-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from Vibrio cholerae (VcMTAN) and Escherichia coli (EcMTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar. However, binding of transition-state analogues is dominated by favorable entropy in VcMTAN and by enthalpy in EcMTAN. Catalytic sites of VcMTAN and EcMTAN in contact with reactants differ by two residues; Ala113 and Val153 in VcMTAN are Pro113 and Ile152, respectively, in EcMTAN. We mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation of altering its properties toward EcMTAN. Inhibition of VcMTAN by transition-state analogues required filling both active sites of the homodimer. However, in the Val153Ile mutant or double mutants, transition-state analogue binding at one site caused complete inhibition. Therefore, a single amino acid, Val153, alters the catalytic site cooperativity in VcMTAN. The transition-state analogue affinity and thermodynamics in mutant VcMTAN became even more unlike those of EcMTAN, the opposite of expectations from catalytic site similarity; thus, catalytic site contacts in VcMTAN are unable to recapitulate the properties of EcMTAN. X-ray crystal structures of EcMTAN, VcMTAN, and a multiple-site mutant of VcMTAN most closely resembling EcMTAN in catalytic site contacts show no major protein conformational differences. The overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences.