Catalytic Residues In Active Site Research Articles

Structure and function of the pseudouridine 5’-monophosphate glycosylase PUMY from Arabidopsis thaliana

ABSTRACT Pseudouridine is a noncanonical C-nucleoside containing a C-C glycosidic linkage between uracil and ribose. In the two-step degradation of pseudouridine, pseudouridine 5’-monophosphate glycosylase (PUMY) is responsible for the second step and catalyses the cleavage of the C-C glycosidic bond in pseudouridine 5’-monophosphate (ΨMP) into uridine and ribose 5’-phosphate, which are recycled via other metabolic pathways. Structural features of Escherichia coli PUMY have been reported, but the details of the substrate specificity of ΨMP were unknown. Here, we present three crystal structures of Arabidopsis thaliana PUMY in different ligation states and a kinetic analysis of ΨMP degradation. The results indicate that Thr149 and Asn308, which are conserved in the PUMY family, are structural determinants for recognizing the nucleobase of ΨMP. The distinct binding modes of ΨMP and ribose 5’-phosphate also suggest that the nucleobase, rather than the phosphate group, of ΨMP dictates the substrate-binding mode. An open-to-close transition of the active site is essential for catalysis, which is mediated by two α-helices, α11 and α12, near the active site. Mutational analysis validates the proposed roles of the active site residues in catalysis. Our structural and functional analyses provide further insight into the enzymatic features of PUMY towards ΨMP.

In silico screening of the phytochemicals present in Clitoria ternatea L. as the inhibitors of snake venom phospholipase A2 (PLA2)

Millions of people suffer from snake bite envenomation, and its management is a challenge, even today. Medicinal plants have attracted the researcher’s attention for their outstanding advantages in treating many diseases, including snake venom poisoning. Clitoria ternatea L, is a plant popularly known for its various pharmacological effects especially, anti-snake venom property. However, the molecular mechanism behind this is poorly understood. It is reported that snake venom PLA2 is an extensively studied toxic factor. This study is meant to screen the compound’s capability to act as inhibitors of the Daboia russelli snake venom PLA2 through molecular docking and dynamics studies. Our results show that among the 27 compounds taken for the study, only Kaempferol showed good interaction profile with the conserved catalytic active site residues, His48 and Asp49. The pharmacophore features of the compound also demonstrate its exact fitting at the binding pocket. Further RMSD, RMSF, Rg, and hydrogen bond analysis confirmed the stable binding of Kaempferol with PLA2 through molecular dynamic simulations for 100 ns. In addition, the MM/PBSA binding free energy calculation of the complex was also affirming the docking results. The binding free energy (BFE) of Kaempferolis better than the reference compound. ADME and Lipinski’s rule of five reveals its drug like properties. Communicated by Ramaswamy H. Sarma

The Effect of Mutations in the TPR and Ankyrin Families of Alpha Solenoid Repeat Proteins.

Protein repeats are short, highly similar peptide motifs that occur several times within a single protein, for example the TPR and Ankyrin repeats. Understanding the role of mutation in these proteins is complicated by the competing facts that 1) the repeats are much more restricted to a set sequence than non-repeat proteins, so mutations should be harmful much more often because there are more residues that are heavily restricted due to the need of the sequence to repeat and 2) the symmetry of the repeats in allows the distribution of functional contributions over a number of residues so that sometimes no specific site is singularly responsible for function (unlike enzymatic active site catalytic residues). To address this issue, we review the effects of mutations in a number of natural repeat proteins from the tetratricopeptide and Ankyrin repeat families. We find that mutations are context dependent. Some mutations are indeed highly disruptive to the function of the protein repeats while mutations in identical positions in other repeats in the same protein have little to no effect on structure or function.

Molecular cloning and biochemical characterization of a NAD-dependent sorbitol dehydrogenase from cold-adapted Pseudomonas mandelii.

Sugar alcohols (polyols) have important roles as nutrients, anti-freezing agents and scavengers of free radicals in cold-adapted bacteria, but the characteristics of polyol dehydrogenases in cold-adapted bacteria remain largely unknown. In this study, based on the observation that a cold-adapted bacterium Pseudomonas mandelii JR-1 predominantly utilized d-sorbitol as its carbon source, among the four polyols examined (d-galactitol, d-mannitol, d-sorbitol and d-xylitol), we cloned and characterized a sorbitol dehydrogenase (SDH, EC 1.1.1.14) belonging to the short-chain dehydrogenase/reductase family from this bacterium (the SDH hereafter referred to as PmSDH). PmSDH contained Asn111, Ser140, Tyr153 and Lys157 as catalytic active site residues and existed as an ∼67-kDa dimer in size-exclusion chromatography. PmSDH converted d-sorbitol to d-fructose using nicotinamide adenine dinucleotide (NAD+) as a cofactor and, vice versa, d-fructose to d-sorbitol using nicotinamide adenine dinucleotide reduced (NADH) as a cofactor. PmSDH maintained its conformational flexibility, secondary and tertiary structures, and thermal stability at 4-25°C. These results indicate that PmSDH, which has a flexible structure and a high catalytic activity at colder temperatures, is well suited to sorbitol utilization in the cold-adapted bacterium P. mandelii JR-1.

Roles of active-site residues in catalysis, substrate binding, cooperativity, and the reaction mechanism of the quinoprotein glycine oxidase

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on kcat and K0.5 for catalysis, K0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.

The effect of ionic liquid on the structure of active site pocket and catalytic activity of a β-glucosidase from Halothermothrix orenii

Abstract Understanding the inhibition of cellulase activity in the presence of ionic liquids (IL) is extremely important towards engineering IL tolerant enzymes for biomass saccharification and subsequent biofuel production. Here we report the all-atom molecular dynamics simulations of a highly active and thermostable GH1 β-glucosidase (EC 3.2.1.21) B8CYA8 from Halothermothrix orenii in the presence of the IL, 1-ethyl-3-methylimidazolium chloride ([EMIM][Cl]). In the presence of IL, we observed a lack of destabilization of the overall protein structure but instead a localized increase in fluctuations of active site pocket residues corresponding to the loop region, gatekeeper region, aglycone binding site, and glycone binding site. The role of specific residues was confirmed by the Principal Component Analysis (PCA) and Chemical Shift Perturbation (CSP) analyses. Since the enzymatic saccharification requires the proper arrangement of catalytic side chains, residue fluctuation within the active site tunnel affects the reaction rate. Indeed, while B8CYA8 exhibits very high IL tolerance, high concentrations of IL inhibit the enzyme, our analyses show that in high concentrations of IL, the IL accumulates at the active site pocket entrance and near the active site catalytic residues to restrict substrate accessibility to the catalytic site and decreases enzyme activity. Unlike the reaction product glucose, the IL does not change the overall tunnel diameter to constrict substrate access. To understand how IL viscosity may affect enzyme behavior, we determined the self-diffusion constant and show that the presence of IL has a significant effect on substrate access to the catalytic site due to the low diffusivity of the substrate in IL. These results highlight the role played by the residues in active site pocket and the IL properties and may guide in the design of better protein and IL pairs for efficient catalysis.

Molecular docking and dynamic simulations of some medicinal plants compounds against SARS-CoV-2: an in silico study

ABSTRACT COVID-19 pandemic has poses urgent health challenge, and this project aims to identify potential inhibitors to combat this virus. We screened 198 bioactive compounds from five selected medicinal plants previously reported to be antiviral against SARS-CoV-2 protease and two co-receptors followed by molecular dynamics simulations. From the screened compounds, Astragalin demonstrated very strong molecular interactions with the molecular docking binding energies −8.5, −8.0, −7.6 kcal/mol for 6LU7, 6LZG, and 6VXX proteins of SARS-CoV-2, respectively. Hydrogen bonding interaction with the active site catalytic residue HIS-41 or CYS-145 of the main protease SARS-CoV-2 was observed. Binding free energies (ΔGbind) from MM-GBSA after 50 ns MD simulations showed that Astragalin has the highest energy of −33.00 and −34.89 kcal/mol in complex with the main protease and spike glycoprotein of SARS-CoV-2, respectively. The study identifies Astragalin as a better inhibitor for the inactivation of COVID-19 and should be pursued as a potential drug candidate for this virus.

Crystal Structure of a GH3 β-Glucosidase from the Thermophilic Fungus Chaetomium thermophilum.

Beta-glucosidases (β-glucosidases) have attracted considerable attention in recent years for use in various biotechnological applications. They are also essential enzymes for lignocellulose degradation in biofuel production. However, cost-effective biomass conversion requires the use of highly efficient enzymes. Thus, the search for new enzymes as better alternatives of the currently available enzyme preparations is highly important. Thermophilic fungi are nowadays considered as a promising source of enzymes with improved stability. Here, the crystal structure of a family GH3 β-glucosidase from the thermophilic fungus Chaetomium thermophilum (CtBGL) was determined at a resolution of 2.99 Å. The structure showed the three-domain architecture found in other β-glucosidases with variations in loops and linker regions. The active site catalytic residues in CtBGL were identified as Asp287 (nucleophile) and Glu517 (acid/base). Structural comparison of CtBGL with Protein Data Bank (PDB)-deposited structures revealed variations among glycosylated Asn residues. The enzyme displayed moderate glycosylation compared to other GH3 family β-glucosidases with similar structure. A new glycosylation site at position Asn504 was identified in CtBGL. Moreover, comparison with respect to several thermostability parameters suggested that glycosylation and charged residues involved in electrostatic interactions may contribute to the stability of the enzyme at elevated temperatures. The reported CtBGL structure provides additional insights into the family GH3 enzymes and could offer new ideas for further improvements in β-glucosidases for more efficient use in biotechnological applications regarding cellulose degradation.

Molecular docking and dynamic studies of crepiside E beta glucopyranoside as an inhibitor of snake venom PLA2.

Alternative treatments from plant-derived small molecules for neutralizing the venom lethality in snake envenomation are prevalent now. Elephantopus scaber, a tropical plant species has been recognized for its various pharmacological activities and especially anti-snake venom property; however, the molecular basis for this property is not understood. It is reported that snake venom PLA2 is a toxic factor with pharmacological effects independent of their catalytic activity. Here we report the inhibition of catalytic property of Cobra and Viper (group I and group II) snake venom PLA2 by the phytocompounds from E. scaber through molecular docking and dynamics studies. Initially, Lipinski's rule, ADMET, and molecular docking studies were carried out. Our results show that among 124 phytocompounds, crepiside E (deacylcynaropicrin-3' beta-glucopyranoside) has shown interactions with the conserved catalytic active site residues, His 48 and Asp 49, in both the PLA2s. Further, molecular dynamic simulations for 60ns confirmed the stability of crepiside E in the active site of PLA2s and were found to be stable throughout the simulation. In order to understand the drug-likeness of crepiside E, pIC50 and MMGBSA scores were correlated by performing a linear regression analysis. Crepiside E was found to have similar chemical features to that of doxycycline, a known PLA2 inhibitor as indicated by a similarity score of 64.15%. Hence, it is concluded that crepiside E beta glucopyranoside present in Elephantopus scaber contributes to neutralizing the snake venom.

Structural explanation for the tunable substrate specificity of an E. coli nucleoside hydrolase: insights from molecular dynamics simulations

Parasitic protozoa rely on nucleoside hydrolases that play key roles in the purine salvage pathway by catalyzing the hydrolytic cleavage of the N-glycosidic bond that connects nucleobases to ribose sugars. Cytidine-uridine nucleoside hydrolase (CU-NH) is generally specific toward pyrimidine nucleosides; however, previous work has shown that replacing two active site residues with Tyr, specifically the Thr223Tyr and Gln227Tyr mutations, allows CU-NH to process inosine. The current study uses molecular dynamics (MD) simulations to gain atomic-level insight into the activity of wild-type and mutant E. coli CU-NH toward inosine. By examining systems that differ in the identity and protonation states of active site catalytic residues, key enzyme-substrate interactions that dictate the substrate specificity of CU-NH are identified. Regardless of the wild-type or mutant CU-NH considered, our calculations suggest that inosine binding is facilitated by interactions of the ribose moiety with active site residues and Ca2+, and π-interactions between two His residues (His82 and His239) and the nucleobase. However, the lack of observed activity toward inosine for wild-type CU-NH is explained by no residue being correctly aligned to stabilize the departing nucleobase. In contrast, a hydrogen-bonding network between hypoxanthine and a newly identified general acid (Asp15) is present when the two Tyr mutations are engineered into the active site. Investigation of the single CU-NH mutants reveals that this hydrogen-bonding network is only maintained when both Tyr mutations are present due to a π-interaction between the residues. These results rationalize previous experiments that show the single Tyr mutants are unable to efficiently hydrolyze inosine and explain how the Tyr residues work synergistically in the double mutant to stabilize the nucleobase leaving group during hydrolysis. Overall, our simulations provide a structural explanation for the substrate specificity of nucleoside hydrolases, which may be used to rationally develop new treatments for kinetoplastid diseases.

In vitro potentiation of carbapenems with tannic acid against carbapenemase-producing enterobacteriaceae: exploring natural products as potential carbapenemase inhibitors

We hypothesized and confirmed that tannic acid (TA) reverses carbapenem resistance by inhibiting carbapenemases in class A and B carbapenemase-producing Enterobacteriaceae. Minimum inhibitory concentrations of carbapenems in the presence and absence of TA and other efflux pump inhibitors, TA-carbapenemases inhibition assays and computational studies showed that TA had the greatest effect on metallo-β-lactamases (MBLs) followed by class A serine-β-lactamases (SBLs). TA completely reversed the MICs of MBL producers from between 32 and ≥512mgl-1 to susceptible values (<4mgl-1 ) while substantially reducing the MICs of SBLs from between 16 and >512mgl-1 to <4 to 16mgl-1 . Tolerable cytotoxic effect was observed for the concentrations tested (8-1024mgl-1 ). TA inhibited enzymes with a marked difference of ≈50% inhibition (IC50 ) for NDM-1 (270μmoll-1 ) and KPC-2 (15 μmoll-1 ). TA inhibited both MBLs and SBLs by targeting their hydrophobic sites. Moreover, TA had a stronger binding affinity for MBLs than SBLs as the MBLs, specifically VIM-1 (-43·7220±0·4513kcalmol-1 ) and NDM-1(-44·2329±0·3806kcalmol-1 ), interact with a larger number of their catalytic active-site residues than that of OXA-48 (-22·5275± 0·1300kcalmol-1 ) and KPC-2 (-22·1164±0·0111kcalmol-1 ). Tannic acid or its analogues could bedeveloped into carbapenemase-inhibiting adjuvants to restore carbapenemactivity in CRE infections, save many lives and reduce healthcare associated costs.

Investigating Amino Acid Residues in The Active Site of Dibenzothiophene Monooxygenase (DszC)

Fossil fuel combustion releases sulfur dioxide into the atmosphere, which is a harmful air pollutant and precursor of sulfate aerosols that contribute to acid rain. It is necessary to reduce atmospheric sulfur levels to mitigate these effects. A current method, hydrodesulfurization, is costly and inefficient at removing sulfur from complex aromatic compounds. Biodesulfurization is a cost‐effective alternative that utilizes bacteria to remove sulfur. Rhodococcus erythropolis uses a four enzyme pathway to remove sulfur from dibenzothiophene. The first enzyme of the pathway, dibenzothiophene monooxygenase (DszC), requires molecular oxygen, reduced flavin, and the formation of an intermediate, C4a‐hydroperoxyflavin, to sequentially oxidize dibenzothiophene to dibenzothiophene sulfone. This study aims to elucidate the roles of DszC active site residues in catalysis and binding and to enable rational engineering of DszC for future application in fuel refinement. Active site residue mutants were created using polymerase chain reaction, followed by plasmid DNA isolation and sequence confirmation. Steady state activity assays of DszC, quantitated by reverse‐phase HPLC, compared the amount of product formed by the mutants relative to wild type‐DszC. The ability of the mutants to form the intermediate was studied using stopped‐flow spectrophotometry. In this study, seven active site residue mutants were created and the percent of product formation by each mutant relative to wild type‐DszC was: His92Asn (12%), His92Gln (20%), Tyr96Phe (12%), His388Ala (38%), His388Asn (39%), His391Val (0%) and Asp392Ala (3%). These results indicate that all of the residues analyzed are necessary for proper product formation. Additionally, the intermediate formation rate constant for Tyr96Phe (1.03 × 104 M−1 s−1) was similar to wild type‐DszC (1.69 × 104 M−1 s−1). These results indicate that Tyr96 is not critical for proper intermediate formation, but has another role in catalysis as evidenced by the low (12%) product formation. His92Asn and Asp392Ala were also analyzed using stopped‐flow spectrophotometry but showed no intermediate formation signifying that these residues are essential to proper intermediate formation. Overall, these results show the necessity of all the residues presented in product formation and the fundamental roles of residues His92 and Asp392 in intermediate formation.Support or Funding InformationNational Institute of Health (NIH SCORE 5SC2AI109500)Research Corporation (CCSA 22672)MBRS/RISE Program/National Institute of Health (Grant 2R25GM063787‐10)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Kinetic and structural analyses reveal residues in phosphoinositide 3-kinase α that are critical for catalysis and substrate recognition

Phosphoinositide 3-kinases (PI3Ks) are ubiquitous lipid kinases that activate signaling cascades controlling cell survival, proliferation, protein synthesis, and vesicle trafficking. PI3Ks have dual kinase specificity: a lipid kinase activity that phosphorylates the 3'-hydroxyl of phosphoinositides and a protein-kinase activity that includes autophosphorylation. Despite the wealth of biochemical and structural information on PI3Kα, little is known about the identity and roles of individual active-site residues in catalysis. To close this gap, we explored the roles of residues of the catalytic domain and the regulatory subunit of human PI3Kα in lipid and protein phosphorylation. Using site-directed mutagenesis, kinetic assays, and quantitative mass spectrometry, we precisely mapped key residues involved in substrate recognition and catalysis by PI3Kα. Our results revealed that Lys-776, located in the P-loop of PI3Kα, is essential for the recognition of lipid and ATP substrates and also plays an important role in PI3Kα autophosphorylation. Replacement of the residues His-936 and His-917 in the activation and catalytic loops, respectively, with alanine dramatically changed PI3Kα kinetics. Although H936A inactivated the lipid kinase activity without affecting autophosphorylation, H917A abolished both the lipid and protein kinase activities of PI3Kα. On the basis of these kinetic and structural analyses, we propose possible mechanistic roles of these critical residues in PI3Kα catalysis.

Intein-Promoted Cyclization of Aspartic Acid Flanking the Intein Leads to Atypical N-Terminal Cleavage.

Protein splicing is a post-translational reaction facilitated by an intein, or intervening protein, which involves the removal of the intein and the ligation of the flanking polypeptides, or exteins. A DNA polymerase II intein from Pyrococcus abyssi (Pab PolII intein) can promote protein splicing in vitro on incubation at high temperature. Mutation of active site residues Cys1, Gln185, and Cys+1 to Ala results in an inactive intein precursor, which cannot promote the steps of splicing, including cleavage of the peptide bond linking the N-extein and intein (N-terminal cleavage). Surprisingly, coupling the inactivating mutations to a change of the residue at the C-terminus of the N-extein (N-1 residue) from the native Asn to Asp reactivates N-terminal cleavage at pH 5. Similar "aspartic acid effects" have been observed in other proteins and peptides but usually only occur at lower pH values. In this case, however, the unusual N-terminal cleavage is abolished by mutations to catalytic active site residues and unfolding of the intein, indicating that this cleavage effect is mediated by the intein active site and the intein fold. We show via mass spectrometry that the reaction proceeds through cyclization of Asp resulting in anhydride formation coupled to peptide bond cleavage. Our results add to the richness of the understanding of the mechanism of protein splicing and provide insight into the stability of proteins at moderately low pH. The results also explain, and may help practitioners avoid, a side reaction that may complicate intein applications in biotechnology.

Discovery and characterization of an F420-dependent glucose-6-phosphate dehydrogenase (Rh-FGD1) from Rhodococcus jostii RHA1

Cofactor F420, a 5-deazaflavin involved in obligatory hydride transfer, is widely distributed among archaeal methanogens and actinomycetes. Owing to the low redox potential of the cofactor, F420-dependent enzymes play a pivotal role in central catabolic pathways and xenobiotic degradation processes in these organisms. A physiologically essential deazaflavoenzyme is the F420-dependent glucose-6-phosphate dehydrogenase (FGD), which catalyzes the reaction F420 + glucose-6-phosphate → F420H2 + 6-phospho-gluconolactone. Thereby, FGDs generate the reduced F420 cofactor required for numerous F420H2-dependent reductases, involved e.g., in the bioreductive activation of the antitubercular prodrugs pretomanid and delamanid. We report here the identification, production, and characterization of three FGDs from Rhodococcus jostii RHA1 (Rh-FGDs), being the first experimental evidence of F420-dependent enzymes in this bacterium. The crystal structure of Rh-FGD1 has also been determined at 1.5 Å resolution, showing a high similarity with FGD from Mycobacterium tuberculosis (Mtb) (Mtb-FGD1). The cofactor-binding pocket and active-site catalytic residues are largely conserved in Rh-FGD1 compared with Mtb-FGD1, except for an extremely flexible insertion region capping the active site at the C-terminal end of the TIM-barrel, which also markedly differs from other structurally related proteins. The role of the three positively charged residues (Lys197, Lys258, and Arg282) constituting the binding site of the substrate phosphate moiety was experimentally corroborated by means of mutagenesis study. The biochemical and structural data presented here provide the first step towards tailoring Rh-FGD1 into a more economical biocatalyst, e.g., an F420-dependent glucose dehydrogenase that requires a cheaper cosubstrate and can better match the demands for the growing applications of F420H2-dependent reductases in industry and bioremediation.

Roles of Conserved Residues of the Glycine Oxidase GoxA in Controlling Activity, Cooperativity, Subunit Composition, and Cysteine Tryptophylquinone Biosynthesis

GoxA is a glycine oxidase that possesses a cysteine tryptophylquinone (CTQ) cofactor that is formed by posttranslational modifications that are catalyzed by a modifying enzyme GoxB. It is the second known tryptophylquinone enzyme to function as an oxidase, the other being the lysine ϵ-oxidase, LodA. All other enzymes containing CTQ or tryptophan tryptophylquinone (TTQ) cofactors are dehydrogenases. Kinetic analysis of GoxA revealed allosteric cooperativity for its glycine substrate, but not O2 This is the first CTQ- or TTQ-dependent enzyme to exhibit cooperativity. Here, we show that cooperativity and homodimer stabilization are strongly dependent on the presence of Phe-237. Conversion of this residue, which is a Tyr in LodA, to Tyr or Ala eliminates the cooperativity and destabilizes the dimer. These mutations also significantly affect the kcat and Km values for the substrates. On the basis of structural and modeling studies, a mechanism by which Phe-237 exerts this influence is presented. Two active site residues, Asp-547 and His-466, were also examined and shown by site-directed mutagenesis to be critical for CTQ biogenesis. This result is compared with the results of similar studies of mutagenesis of structurally conserved residues of other tryptophylquinone enzymes. These results provide insight into the roles of specific active-site residues in catalysis and CTQ biogenesis, as well as describing an interesting mechanism by which a single residue can dictate whether or not an enzyme exhibits cooperative allosteric behavior toward a substrate.

The conserved active site histidine-glutamate pair of ferrochelatase coordinately catalyzes porphyrin metalation

Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to generate heme. Despite recent research on the reaction mechanism of ferrochelatase, the precise roles and localization of individual active site residues in catalysis, particularly those involved in the insertion of the ferrous iron into the protoporphyrin IX substrate, remain controversial. One outstanding question is from which side of the macrocycle of the bound porphyin substrate is the ferrous iron substrate inserted. Pre-steady state kinetic experiments done under single-turnover conditions conclusively demonstrate that metal ion insertion is pH-dependent, and that the conserved active site His-Glu pair coordinately catalyzes the metal ion insertion reaction. Further, p[Formula: see text] calculations and molecular dynamic simulations indicate that the active site His is deprotonated and the protonation state of the Glu relates to the conformational state of ferrochelatase. Specifically, the conserved Glu in the open conformation of ferrochelatase is deprotonated, while it remains protonated in the closed conformation. These findings support not only the role of the His-Glu pair in catalyzing metal ion insertion, as these residues need to be deprotonated to bind the incoming metal ion, but also the importance of the relationship between the protonation state of the Glu residue and the conformation of ferrochelatase. Finally, the results of this study are consistent with our previous proposal that the unwinding of the [Formula: see text]-helix, the major structural determinant of the closed to open conformational transition in ferrochelatase, is associated with the Glu residue binding the Fe[Formula: see text] substrate from a mitochondrial Fe[Formula: see text] donor.

The inhibition of human farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates. Elucidating the role of active site threonine 201 and tyrosine 204 residues using enzyme mutants

Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins.Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs.In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding.

The Integration and Excision of CTnDOT.

Bacteroides species are one of the most prevalent groups of bacteria present in the human colon. Many strains carry large, integrated elements including integrative and conjugative elements (ICEs). One such ICE is CTnDOT, which is 65 kb in size and encodes resistances to tetracycline and erythromycin. CTnDOT has been increasing in prevalence in Bacteroides spp., and is now found in greater than 80% of natural isolates. In recent years, CTnDOT has been implicated in the spread of antibiotic resistance among gut microbiota. Interestingly, the excision and transfer of CTnDOT is stimulated in the presence of tetracycline. The tyrosine recombinase IntDOT catalyzes the integration and excision reactions of CTnDOT. Unlike the well-characterized lambda Int, IntDOT tolerates heterology in the overlap region between the sites of cleavage and strand exchange. IntDOT also appears to have a different arrangement of active site catalytic residues. It is missing one of the arginine residues that is conserved in other tyrosine recombinases. The excision reaction of CTnDOT is complex, involving excision proteins Xis2c, Xis2d, and Exc, as well as IntDOT and a Bacteroides host factor. Xis2c and Xis2d are small, basic proteins like other recombination directionality factors (RDFs). Exc is a topoisomerase; however, the topoisomerase function is not required for the excision reaction. Exc has been shown to stimulate excision frequencies when there are mismatches in the overlap regions, suggesting that it may play a role in resolving Holliday junctions (HJs) containing heterology. Work is currently under way to elucidate the complex interactions involved with the formation of the CTnDOT excisive intasomes.

Structure-Based Enzyme Tailoring of 5-Hydroxymethylfurfural Oxidase

5-Hydroxymethylfurfural oxidase (HMFO) is a flavin-dependent enzyme that catalyzes the oxidation of many aldehydes, primary alcohols, and thiols. The three-step conversion of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid is relevant for the industrial production of biobased polymers. The remarkable wide substrate scope of HMFO contrasts with the enzyme’s precision in positioning the substrate to perform catalysis. We have solved the crystal structure of HMFO at 1.6 A resolution, which guided mutagenesis experiments to probe the role of the active-site residues in catalysis. Mutations targeting two active-site residues generated engineered forms of HMFO with promising catalytic features, namely enantioselective activities on secondary alcohols and improved 2,5-furandicarboxylic acid yields.