Effects Of Active Site Mutations Research Articles

Expanding the Substrate Scope of a Bacterial Nucleotidyltransferase via Allosteric Mutations.

Bacterial glycoconjugates, such as cell surface polysaccharides and glycoproteins, play important roles in cellular interactions and survival. Enzymes called nucleotidyltransferases use sugar-1-phosphates and nucleoside triphosphates (NTPs) to produce nucleoside diphosphate sugars (NDP-sugars), which serve as building blocks for most glycoconjugates. Research spanning several decades has shown that some bacterial nucleotidyltransferases have broad substrate tolerance and can be exploited to produce a variety of NDP-sugars in vitro. While these enzymes are known to be allosterically regulated by NDP-sugars and their fragments, much work has focused on the effect of active site mutations alone. Here, we show that rational mutations in the allosteric site of the nucleotidyltransferase RmlA lead to expanded substrate tolerance and improvements in catalytic activity that can be explained by subtle changes in quaternary structure and interactions with ligands. These observations will help inform future studies on the directed biosynthesis of diverse bacterial NDP-sugars and downstream glycoconjugates.

Investigation new positions for catalytic activity of Chaetomium thermophilum and Ceriporiopsis subvermispora formate dehydrogenases

NAD+-dependent formate dehydrogenases (FDHs, E.C 1.2.1.2) catalyse the reversible reaction of CO2 to formate ion (HCOO−) and reduces NAD+ molecule to NADH. Previously described FDHs from Chaetomium thermophilum (CtFDH) and Ceriporiopsis subvermispora (CsFDH) are active against formate and HCO3 –. In this study, we examined the functional effects of active site mutations in Ct and Cs NAD+-dependent FDHs. The residues Ile94, Asn120, Val310, His312 at CtFDH and Asn312, Val313, Val331 at CsFDH are located in the active site. The effects of amino acid changes on catalytic properties and thermal stability of CtFDH and CsFDH revealed some interesting results compared with structurally equivalent positions that have been studied in the literature. The strongest effect was observed in CsFDH Val313Pro against HCO3 –. The KM value of the CsFDH Val313 enzyme for HCO3 – substrate dramatically decreased, and enzyme activity increased. In CtFDH mutants, all enzymes except Val310Asn showed an increased kcat value when KM values increase. Analyses of results of mutant CtFDHs and CsFDHs give some promising results for CO2 reduction as compared to the literature. Structural analyses of the substrate-binding site were done by homology modelling.

Structural insights into the effect of active-site mutation on the catalytic mechanism of carbonic anhydrase.

Enzymes are catalysts of biological processes. Significant insight into their catalytic mechanisms has been obtained by relating site-directed mutagenesis studies to kinetic activity assays. However, revealing the detailed relationship between structural modifications and functional changes remains challenging owing to the lack of information on reaction intermediates and of a systematic way of connecting them to the measured kinetic parameters. Here, a systematic approach to investigate the effect of an active-site-residue mutation on a model enzyme, human carbonic anhydrase II (CA II), is described. Firstly, structural analysis is performed on the crystallographic intermediate states of native CA II and its V143I variant. The structural comparison shows that the binding affinities and configurations of the substrate (CO2) and product (HCO3 -) are altered in the V143I variant and the water network in the water-replenishment pathway is restructured, while the proton-transfer pathway remains mostly unaffected. This structural information is then used to estimate the modifications of the reaction rate constants and the corresponding free-energy profiles of CA II catalysis. Finally, the obtained results are used to reveal the effect of the V143I mutation on the measured kinetic parameters (k cat and k cat/K m) at the atomic level. It is believed that the systematic approach outlined in this study may be used as a template to unravel the structure-function relationships of many other biologically important enzymes.

Functional effects of active site mutations in NAD+-dependent formate dehydrogenases on transformation of hydrogen carbonate to formate.

Conversion of hydrogen carbonate to formate by mutants of Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) formate dehydrogenases (FDHs) was studied. Hydrogen carbonate is not the primary substrate for the hydride transfer reaction in FDHs. The chosen mutations were selected so that enzyme activity could remain at an adequate level. In CtFDH, the mutation Asn120Cys in the active site inactivated the enzyme for formate (oxidation) but increased the specific activity for hydrogen carbonate (reduction) as a function of substrate concentration. The mutation Asn120Cys in CtFDH increased 6.5-fold the KM, indicating that substrate binding was weakened. A 6.5-fold increase of kcat compensated the lower affinity suggesting that product release was improved. The corresponding mutation Asn119Cys in CmFDH inactivated the enzyme for both substrates. Molecular dynamics simulations indicated that the active site dimensions change differently with different substrates after mutations, and in the mutant Asn120Cys of CtFDH, hydrogen carbonate adopted better reactive position than formate. With hydrogen carbonate, the active site enlarged enough for two hydrogen carbonate molecules to be placed there. The change of Asn119 to bulky Tyr or His in CmFDH requires changes in the active site to accommodate the substrate; activity with formate was retained but not with hydrogen carbonate. This study showed that the active site of FDHs can be modified radically, which gives possibilities for further enzyme engineering to improve the reaction with hydrogen carbonate or carbon dioxide for enzymatic fixing of carbon dioxide.

Insights from molecular dynamics on substrate binding and effects of active site mutations in Δ1-pyrroline-5-carboxylate dehydrogenase

The NAD+-dependent enzyme, Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH), has an important role in proline and hydroxyproline catabolism for humans. Specifically, this aldehyde dehydrogenase is responsible for the oxidation of both l-glutamate-γ-semialdehyde (GSA) and 4-erythro-hydroxy-l-glutamate-γ-semialdehyde (4-OH-GSA) to their respective l-glutamate product forms. We have performed a detailed molecular dynamics (MD) study of both the reactant and product complex structures of P5CDH to gain insights into ligand binding (i.e., GSA, 4-OH-GSA, NAD+, GLU) in the active site. Moreover, our investigations were further extended to examine the structural impact of S352L, S352A, and E314A mutations on the deficiency in the P5CDH enzymatic activity. Our in silico mutation analysis indicated that the conserved Glu447 has significantly shifted in both the S352L and E314A mutants, causing NAD+ to be displaced from its predictive orientation in the binding site and hence forming a catalytically inactive enzyme. However in the case of S352A, the catalytic site including the oxyanion hole and Cys348 remain virtually unchanged, and the coenzyme maintains its binding position.

Effects of Active Site Mutations on Specificity of Nucleobase Binding in Human DNA Polymerase η.

Human DNA polymerase η (Pol η) plays a vital role in protection against skin cancer caused by damage from ultraviolet light. This enzyme rescues stalled replication forks at cyclobutane thymine–thymine dimers (TTDs) by inserting nucleotides opposite these DNA lesions. Residue R61 is conserved in the Pol η enzymes across species, but the corresponding residue, as well as its neighbor S62, is different in other Y-family polymerases, Pol ι and Pol κ. Herein, R61 and S62 are mutated to their Pol ι and Pol κ counterparts. Relative binding free energies of dATP to mutant Pol η•DNA complexes with and without a TTD were calculated using thermodynamic integration. The binding free energies of dATP to the Pol η•DNA complex with and without a TTD are more similar for all of these mutants than for wild-type Pol η, suggesting that these mutations decrease the ability of this enzyme to distinguish between a TTD lesion and undamaged DNA. Molecular dynamics simulations of the mutant systems provide insights into the molecular level basis for the changes in relative binding free energies. The simulations identified differences in hydrogen-bonding, cation−π, and π–π interactions of the side chains with the dATP and the TTD or thymine–thymine (TT) motif. The simulations also revealed that R61 and Q38 act as a clamp to position the dATP and the TTD or TT and that the mutations impact the balance among the interactions related to this clamp. Overall, these calculations suggest that R61 and S62 play key roles in the specificity and effectiveness of Pol η for bypassing TTD lesions during DNA replication. Understanding the basis for this specificity is important for designing drugs aimed at cancer treatment.

Application of resonance Raman spectroscopy for interrogation of cryoradiolytically reduced oxygenated heme proteins

Resonance Raman (rR) spectroscopy is generally useful to interrogate unstable intermediates that arise in the enzymatic cycles of heme enzymes, such as peroxo-, hydroperoxo- and ferryl species. While it is typically difficult to trap these elusive intermediates in solution, the cryoradiolysis approach offers a way to successfully overcome these obstacles. The present work employs modified derivatives of horse heart myoglobin to demonstrate the methodology and utility of a combination of rR and cryoradiolysis for detection and structural characterization of Fe―O―O, Fe―O―O―H and FeO fragments of such reactive species. First, the effect of active site mutations on the Fe―O―O fragments was investigated; specifically, the His64Leu, Val68Ser and Ile107Ala in the distal pocket and Ser92Ala, Ser92Leu and His97Phe on the proximal side. The current work has permitted documentation of the wavenumbers of the ν(Fe―O) stretching modes of the relatively unstable dioxygen adducts of these mutants, some of them previously unreported. Significantly, applying the cryoradiolysis approach has yielded previously unavailable vibrational spectroscopic data for the peroxo-, hydroperoxo and ferryl derivatives of these mutants. Of the six mutations investigated, only two, the H64L and V68S, replacements had a significant effect on the structure and stability of these unstable intermediates. In addition to these manipulations of the heme active site environment, the study of myoglobins bearing modified hemes was also undertaken; namely, myoglobins reconstituted with mesoheme and diformyl deuteroheme. The results show that the heme equatorial electronic effects significantly influence the stability and internal modes of the Fe―O―O and FeO fragments of these unstable oxy intermediates. Copyright © 2016 John Wiley & Sons, Ltd.

Effect of active site mutation on pH activity and transglycosylation of Sulfolobus acidocaldarius β-glycosidase

Sulfolobus acidocaldarius β-glycosidase (BGAL_SULAC) was used as an extremophilic enzyme model to study the effect of mutations close to the catalytic residues on the enzyme activity and the pH-activity profile. We report here the results for three mutations (N211D, V212D and V212T) changing the polarity close to the putative acid/base catalyst E209. N211D was outside the H-bonding distance from E209, whereas V212D and V212T were in H-bonding distance from E209. V212D and V212T shifted the pH-activity profile towards acidic pH with both lactose and cellobiose as substrates. N211D and V212D decreased clearly the activity. Although V212T increased 6-fold the Km value with cellobiose, the mutant showed higher specific activity in high substrate concentrations. The reason was greatly reduced production of trisaccharide by V212T from cellobiose by transglycosylation. Threonine differs by the terminal oxygen from valine, indicating that additional hydrogen bonding to substrate or reaction products may affect the reaction behavior of the enzyme. Although the mutations in the active site are often harmful, the mutation V212T showed biotechnologically promising properties.

Structures and activities of archaeal members of the LigD 3′-phosphoesterase DNA repair enzyme superfamily

LigD 3′-phosphoesterase (PE) is a component of the bacterial NHEJ apparatus that performs 3′-end-healing reactions at DNA breaks. The tertiary structure, active site and substrate specificity of bacterial PE are unique vis–à-vis other end-healing enzymes. PE homologs are present in archaea, but their properties are uncharted. Here, we demonstrate the end-healing activities of two archaeal PEs—Candidatus Korarchaeum cryptofilum PE (CkoPE; 117 amino acids) and Methanosarcina barkeri PE (MbaPE; 151 amino acids)—and we report their atomic structures at 1.1 and 2.1 Å, respectively. Archaeal PEs are minimized versions of bacterial PE, consisting of an eight-stranded β barrel and a 310 helix. Their active sites are located in a crescent-shaped groove on the barrel’s outer surface, wherein two histidines and an aspartate coordinate manganese in an octahedral complex that includes two waters and a phosphate anion. The phosphate is in turn coordinated by arginine and histidine side chains. The conservation of active site architecture in bacterial and archaeal PEs, and the concordant effects of active site mutations, underscore a common catalytic mechanism, entailing transition state stabilization by manganese and the phosphate-binding arginine and histidine. Our results fortify the proposal that PEs comprise a DNA repair superfamily distributed widely among taxa.

Effect of Active-site Mutation at Asn67 on the Proton Transfer Mechanism of Human Carbonic Anhydrase II

The rate-limiting proton transfer (PT) event in the site-specific mutant N67L of human carbonic anhydrase II (HCA II) has been examined by kinetic, X-ray, and simulation approaches. The X-ray crystallography studies, which were previously reported, and molecular dynamics (MD) simulations indicate that the proton shuttling residue, His64, predominantly resides in the outward orientation with a significant disruption of the ordered water in the active site for the dehydration pathway. While disorder is seen in the active-site water, water cluster analysis indicates that the N67L mutant may form water clusters similar to those seen in the wild-type (WT). For the hydration pathway of the enzyme, the active site water cluster analysis reveals an inability of the N67L mutant to stabilize water clusters when His64 is in the inward orientation, thereby favoring PT when His64 is in the outward orientation. The preference of the N67L mutant to carry out the PT when His64 is in the outward orientation for both the hydration and dehydration pathway is reasoned to be the main cause of the observed reduction in the overall rate. To probe the mechanism of PT, solvent H/D kinetic isotope effects (KIEs) were experimentally studied with catalysis measured by the exchange of (18)O between CO(2) and water. The values obtained from the KIEs were determined as a function of the deuterium content of solvent, using the proton inventory method. No differences were detected in the overarching mechanism of PT between WT and N67L HCA II, despite changes in the active-site water structure and/or the orientation of His64.

Effects of active site mutations in haemoglobin I from Lucina pectinata: a molecular dynamic study

Haemoglobin I from Lucina pectinata is a monomeric protein consisting of 142 amino acids. Its active site contains a peculiar arrangement of phenylalanine residues (PheB10, PheCD1 and PheE11) and a distal Gln at position E7. Active site mutations at positions B10, E7 and E11 were performed in deoxy haemoglobin I (HbI), followed by 10 ns molecular dynamic simulations. The results showed that the mutations induced changes in domains far from the active site producing more flexible structures than the native HbI. Distance analyses revealed that the heme pocket amino acids at positions E7 and B10 are extremely sensitive to any heme pocket residue mutation. The high flexibility observed by the E7 position suggests an important role in the ligand binding kinetics in ferrous HbI, while both positions play a major role in the ligand stabilisation processes. Furthermore, our results showed that E11Phe plays a pivotal role in protein stability.

Dissecting Tn5 Transposition Using HIV-1 Integrase Diketoacid Inhibitors

Diketoacid (DKA) compounds have been shown to inhibit HIV-1 integrase by a mechanism that involves sequestration of the active site metals. Because HIV-1 integrase and Tn5 transposase have similar active site architectures and catalytic mechanisms, we investigated whether DKA analogues would inhibit Tn5 transposase activity and provide a model system to explore the mechanisms of action of these inhibitors. A screen of several hundred DKA analogues identified several with activity against Tn5 Tnp. Six DKA inhibitors used in this study manifested a variety of effects on different transposition steps suggesting that different analogues may have different binding contacts with transposase. All DKA compounds inhibited paired end complex (PEC) formation in which the nucleoprotein complex required for catalysis is assembled. Dissociation of PECs by some DKA compounds indicates that these inhibitors can decrease PEC stability. Four DKA compounds inhibited the two cleavage steps releasing transposon DNA from flanking DNA, and one of these four compounds preferentially inhibited the second cleavage step. The differential effect of this inhibitor on the second cleavage event indicates that cleavage of the two transposon-donor DNA boundaries is a sequential process requiring a conformational change. The requirement for a conformational change between cleavage events was also demonstrated by the inability of transposase to perform second cleavage at 25 degrees C. Finally, all six compounds inhibit strand transfer, the final step of Tn5 transposition. Two of the compounds that inhibited strand transfer have no effect on DNA cleavage. The strand transfer inhibition properties of various DKA compounds was sensitive to the structure of the 5'-non-transferred strand, suggesting that these compounds bind in or near the transposase active site. Other results that probe compound binding sites include the effects of active site mutations and donor DNA on DKA compound inhibition activities. Thus, DKA inhibitors will provide an important set of tools to investigate the mechanism of action of transposases and integrases.

Structural basis for interaction of O-acetylserine sulfhydrylase and serine acetyltransferase in the Arabidopsis cysteine synthase complex.

In plants, association of O-acetylserine sulfhydrylase (OASS) and Ser acetyltransferase (SAT) into the Cys synthase complex plays a regulatory role in sulfur assimilation and Cys biosynthesis. We determined the crystal structure of Arabidopsis thaliana OASS (At-OASS) bound with a peptide corresponding to the C-terminal 10 residues of Arabidopsis SAT (C10 peptide) at 2.9-A resolution. Hydrogen bonding interactions with key active site residues (Thr-74, Ser-75, and Gln-147) lock the C10 peptide in the binding site. C10 peptide binding blocks access to OASS catalytic residues, explaining how complex formation downregulates OASS activity. Comparison with bacterial OASS suggests that structural plasticity in the active site allows binding of SAT C termini with dissimilar sequences at structurally similar OASS active sites. Calorimetric analysis of the effect of active site mutations (T74S, S75A, S75T, and Q147A) demonstrates that these residues are important for C10 peptide binding and that changes at these positions disrupt communication between active sites in the homodimeric enzyme. We also demonstrate that the C-terminal Ile of the C10 peptide is required for molecular recognition by At-OASS. These results provide new insights into the molecular mechanism underlying formation of the Cys synthase complex and provide a structural basis for the biochemical regulation of Cys biosynthesis in plants.

Effects of Active Site Mutations on the Metal Binding Affinity, Catalytic Competence, and Stability of the Family II Pyrophosphatase fromBacillus subtilis

Family II inorganic pyrophosphatases (PPases) have been recently found in a variety of bacteria. Their primary and tertiary structures differ from those of the well-known family I PPases, although both have a binuclear metal center directly involved in catalysis. Here, we examined the effects of mutating one Glu, four His, and five Asp residues forming or close to the metal center on Mn(2+) binding affinity, catalysis, oligomeric structure, and thermostability of the family II PPase from Bacillus subtilis (bsPPase). Mutations H9Q, D13E, D15E, and D75E in two metal-binding subsites caused profound (10(4)- to 10(6)-fold) reductions in the binding affinity for Mn(2+). Most of the mutations decreased k(cat) for MgPP(i) by 2-3 orders of magnitude when measured with Mn(2+) or Mg(2+) bound to the high-affinity subsite and Mg(2+) bound to both the low-affinity subsite and pyrophosphate. In the E78D variant, the k(cat) for the Mn-bound enzyme was decreased 120-fold, converting bsPPase from an Mn-specific to an Mg-specific enzyme. K(m) values were less affected by the mutations, and, interestingly, were decreased in most cases. Mutations of His(97) and His(98) residues, which lie near the subunit interface, greatly destabilized the bsPPase dimer, whereas most other mutations stabilized it. Mn(2+), in sharp contrast to Mg(2+), conferred high thermostability to wild-type bsPPase, although this effect was reduced by all of the mutations except D203E. These results indicate that family II PPases have a more integrated active site structure than family I PPases and are consequently more sensitive to conservative mutations.

A major role for a set of non-active site mutations in the development of HIV-1 protease drug resistance.

A major problem in the chemotherapy of HIV-1 infection is the appearance of drug resistance. In the case of HIV-1 protease inhibitors, resistance originates from mutations in the protease molecule that lower the affinity of inhibitors while still maintaining a viable enzymatic profile. Drug resistance mutations can be classified as active site or non-active site mutations depending on their location within the protease molecule. Active site mutations directly affect drug/target interactions, and their action can be readily understood in structural terms. Non-active site mutations influence binding from distal locations, and their mechanism of action is not immediately apparent. In this paper, we have characterized a mutant form of the HIV-1 protease, ANAM-11, identified in clinical isolates from HIV-1 infected patients treated with protease inhibitors. This mutant protease contains 11 mutations, 10 of which are located outside the active site (L10I/M36I/S37D/M46I/R57K/L63P/A71V/G73S/L90M/I93L) and 1 within the active site (I84V). ANAM-11 lowers the binding affinity of indinavir, nelfinavir, saquinavir, and ritonavir by factors of 4000, 3300, 5800, and 80000, respectively. Surprisingly, most of the loss in inhibitor affinity is due to the non-active site mutations as demonstrated by additional experiments performed with a protease containing only the 10 non-active site mutations (NAM-10) and another containing only the active site mutation (A-1). Kinetic analysis with two different substrates yielded comparable catalytic efficiencies for A-1, ANAM-11, NAM-10, and the wild-type protease. These studies demonstrate that non-active site mutations can be the primary source of resistance and that their role is not necessarily limited to compensate deleterious effects of active site mutations. Analysis of the structural stability of the proteases by differential scanning calorimetry reveals that ANAM-11 and NAM-10 are structurally more stable than the wild-type protease while A-1 is less stable. Together, the binding and structural thermodynamic results suggest that the non-active site mutants affect inhibitor binding by altering the geometry of the binding site cavity through the accumulation of mutations within the core of the protease molecule.

Ligand binding sites in Escherichia coli inorganic pyrophosphatase: effects of active site mutations.

Type I soluble inorganic pyrophosphatases (PPases) are well characterized both structurally and mechanistically. Earlier we measured the effects of active site substitutions on pH--rate profiles for the type I PPases from both Escherichia coli (E-PPase) and Saccharomyces cerevisae (Y-PPase). Here we extend these studies by measuring the effects of such substitutions on the more discrete steps of ligand binding to E-PPase, including (a) Mg(2+) and Mn(2+) binding in the absence of added ligand; (b) Mg(2+) binding in the presence of either P(i) or hydroxymethylbisphosphonate (HMBP), a competitive inhibitor of E-PPase; and (c) P(i) binding in the presence of Mn(2+). The active site of a type I PPase has well-defined subsites for the binding of four divalent metal ions (M1--M4) and two phosphates (P1, P2). Our results, considered in light of pertinent results from crystallographic studies on both E-PPase and Y-PPase and parallel functional studies on Y-PPase, allow us to conclude the following: (a) residues E20, D65, D70, and K142 play key roles in the functional organization of the active site; (b) the major structural differences between the product and substrate complexes of E-PPase are concentrated in the lower half of the active site; (c) the M1 subsite is functionally isolated from the rest of the active site; and (d) the M4 subsite is an especially unconstrained part of the active site.

Effect of sequences of the active-site dipeptides of DsbA and DsbC on in vivo folding of multidisulfide proteins in Escherichia coli.

We have examined the role of the active-site CXXC central dipeptides of DsbA and DsbC in disulfide bond formation and isomerization in the Escherichia coli periplasm. DsbA active-site mutants with a wide range of redox potentials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsbA promoter by integration of the respective alleles into the bacterial chromosome. The dsbA alleles gave significant differences in the yield of active murine urokinase, a protein containing 12 disulfides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA. No direct correlation between the in vitro redox potential of dsbA variants and the urokinase yield was observed. These results suggest that the active-site CXXC motif of DsbA can play an important role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential. However, under aerobic conditions, there was no significant difference among the DsbA mutants with respect to phenotypes depending on the oxidation of proteins with few disulfide bonds. The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined. A library of DsbC expression plasmids with the active-site dipeptide randomized was screened for mutants that have increased disulfide isomerization activity. A number of DsbC mutants that showed enhanced expression of a variant of human tissue plasminogen activator as well as mouse urokinase were obtained. These DsbC mutants overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse. Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial periplasmic space.

Effects of active site mutations on the function, co-ordination state and electronic structure of cytochrome c peroxidase

Effects of active site mutations on the function, co-ordination state and electronic structure of cytochrome c peroxidase