An alternate sucrose binding mode in the E203Q Arabidopsis invertase mutant: an X-ray crystallography and docking study

In the present study, we report on the X-ray crystallographic structure of a GH32 invertase mutant, (i.e., the Arabidopsis thaliana cell-wall invertase 1-E203Q, AtcwINV1-mutant) in complex with sucrose. This structure was solved to reveal the features of sugar binding in the catalytic pocket. However, as demonstrated by the X-ray structure the sugar binding and the catalytic pocket arrangement is significantly altered as compared with what was expected based on previous X-ray structures on GH-J clan enzymes. We performed a series of docking and molecular dynamics simulations on various derivatives of AtcwINV1 to reveal the reasons behind this modified sugar binding. Our results demonstrate that the E203Q mutation introduced into the catalytic pocket triggers conformational changes that alter the wild type substrate binding. In addition, this study also reveals the putative productive sucrose binding modus in the wild type enzyme.     

Solution structure of the catalytic domain of human collagenase-3 (MMP-13) complexed to a potent non-peptidic sulfonamide inhibitor: binding comparison with stromelysin-1 and collagenase-1

The full three-dimensional structure of the catalytic domain of human collagenase-3 (MMP-13) complexed to a potent, sulfonamide hydroxamic acid inhibitor (CGS 27023) has been determined by NMR spectroscopy. The results reveal a core domain for the protein consisting of three alpha-helices and five beta-sheet strands with an overall tertiary fold similar to the catalytic domains of other matrix metalloproteinase family members. The S1' pocket, which is the major site of hydrophobic binding interaction, was found to be a wide cleft spanning the length of the protein and presenting facile opportunity for inhibitor extension deep into the pocket. Comparison with the reported X-ray structure of collagenase-3 showed evidence of flexibility for the loop region flanking the S1' pocket in both NMR and X-ray data. This flexibility was corroborated by NMR dynamics studies. Inhibitor binding placed the methoxy phenyl ring in the S1' pocket with the remainder of the molecule primarily solvent-exposed. The binding mode for this inhibitor was found to be similar with respect to stromelysin-1 and collagenase-1; however, subtle comparative differences in the interactions between inhibitor and enzyme were observed for the three MMPs that were consistent with their respective binding potencies.     

Probing flexibility and "induced-fit" phenomena in aldose reductase by comparative crystal structure analysis and molecular dynamics simulations

Aldose reductase is a promising target for the treatment of diabetic complications, and as such, has become the focus of various drug design projects. As revealed by a survey of available crystal structures, the protein shows pronounced induced-fit effects upon ligand binding. Although helping to explain the enzyme's substrate promiscuity, phenomena of this kind are still responsible for significant complications in structure-based design efforts directed to aldose reductase. Accordingly, a deeper understanding of the principles governing conformational alterations in this enzyme would be of utmost practical importance. As a first step in addressing this issue, molecular dynamics (MD) simulations have been carried out. The ultrahigh resolution crystal structure of aldose reductase complexed with inhibitor IDD594 served as ideal starting point for a set of different simulations of nanosecond time scale: the native complexed state with bound inhibitor, the uncomplexed state (after removal of the inhibitor) at standard temperature, and the uncomplexed state at elevated temperature. The reference simulation of the complex exhibits extraordinary stability of the overall fold, whereas two distinct conformational substates are found for the binding-site region. In contrast, already at standard temperature pronounced changes are observed in the binding region during the simulation of the uncomplexed state. Leu300, for example, closes the access to the pocket opened by IDD594. On the other hand, conformations around the catalytic site are highly conserved, with the His110-Tyr48-NADP+ orientation being stabilized by a water molecule. Detailed analysis of the trajectories allows to reveal a set of distinct conformational substates that may prove useful as alternative structural templates in virtual screening for new aldose reductase inhibitors.     

Altering the mouth of a hydrophobic pocket. Structure and kinetics of human carbonic anhydrase II mutants at residue Val-121

Eleven amino acid substitutions at Val-121 of human carbonic anhydrase II including Gly, Ala, Ser, Leu, Ile, Lys, and Arg, were constructed by site-directed mutagenesis. This residue is at the mouth of the hydrophobic pocket in the enzyme active site. The CO2 hydrase activity and the p-nitrophenyl esterase activity of these CAII variants correlate with the hydrophobicity of the residue, suggesting that the hydrophobic character of this residue is important for catalysis. The effects of these mutations on the steady-state kinetics for CO2 hydration occur mainly in kcat/Km and Km, consistent with involvement of this residue in CO2 association. The Val-121----Ala mutant, which exhibits about one-third normal CO2 hydrase activity, has been studied by x-ray crystallographic methods. No significant changes in the mutant enzyme conformation are evident relative to the wild-type enzyme. Since Val-121 is at the mouth of the hydrophobic pocket, its substitution by the methyl side chain of alanine makes the pocket mouth significantly wider than that of the wild-type enzyme. Hence, although a moderately wide (and deep) pocket is important for substrate association, a wider mouth to this pocket does not seriously compromise the catalytic approach of CO2 toward nucleophilic zinc-bound hydroxide.     

Crystal structure of 7,8-dihydropteroate synthase from Bacillus anthracis: mechanism and novel inhibitor design

Dihydropterate synthase (DHPS) is the target for the sulfonamide class of antibiotics, but increasing resistance has encouraged the development of new therapeutic agents against this enzyme. One approach is to identify molecules that occupy the pterin binding pocket which is distinct from the pABA binding pocket that binds sulfonamides. Toward this goal, we present five crystal structures of DHPS from Bacillus anthracis, a well-documented bioterrorism agent. Three DHPS structures are already known, but our B. anthracis structures provide new insights into the enzyme mechanism. We show how an arginine side chain mimics the pterin ring in binding within the pterin binding pocket. The structures of two substrate analog complexes and the first structure of a DHPS-product complex offer new insights into the catalytic mechanism and the architecture of the pABA binding pocket. Finally, as an initial step in the development of pterin-based inhibitors, we present the structure of DHPS complexed with 5-nitro-6-methylamino-isocytosine.     

Structural and functional consequences of coenzyme binding to the inactive asian variant of mitochondrial aldehyde dehydrogenase: roles of residues 475 and 487

The common mitochondrial aldehyde dehydrogenase (ALDH2) ALDH2(*)2 polymorphism is associated with impaired ethanol metabolism and decreased efficacy of nitroglycerin treatment. These physiological effects are due to the substitution of Lys for Glu-487 that reduces the k(cat) for these processes and increases the K(m) for NAD(+), as compared with ALDH2. In this study, we sought to understand the nature of the interactions that give rise to the loss of structural integrity and low activity in ALDH2(*)2 even when complexed with coenzyme. Consequently, we have solved the crystal structure of ALDH2(*)2 complexed with coenzyme to 2.5A(.) We have also solved the structures of a mutated form of ALDH2 where Arg-475 is replaced by Gln (R475Q). The structural and functional properties of the R475Q enzyme are intermediate between those of wild-type and the ALDH2(*)2 enzymes. In both cases, the binding of coenzyme restores most of the structural deficits observed in the apoenzyme structures. The binding of coenzyme to the R475Q enzyme restores its structure and catalytic properties to near wild-type levels. In contrast, the disordered helix within the coenzyme binding pocket of ALDH2(*)2 is reordered, but the active site is only partially reordered. Consistent with the structural data, ALDH2(*)2 showed a concentration-dependent increase in esterase activity and nitroglycerin reductase activity upon addition of coenzyme, but the levels of activity do not approach those of the wild-type enzyme or that of the R475Q enzyme. The data presented shows that Glu-487 maintains a critical function in linking the structure of the coenzyme-binding site to that of the active site through its interactions with Arg-264 and Arg-475, and in doing so, creates the stable structural scaffold conducive to catalysis.     

Interaction of tyrosyl aryl dipeptides with S. aureus tyrosyl tRNA synthetase: inhibition and crystal structure of a complex.

Tyrosyl aryl dipeptide inhibitors of S. aureus tyrosyl tRNA synthetase have been identified with IC50 values down to 0.5 microM. A crystal structure of the enzyme complexed to one of the inhibitors shows occupancy of the tyrosyl binding pocket coupled with inhibitor interactions to key catalytic residues.     

Acetobacter turbidans alpha-amino acid ester hydrolase: how a single mutation improves an antibiotic-producing enzyme

The alpha-amino acid ester hydrolase (AEH) from Acetobacter turbidans is a bacterial enzyme catalyzing the hydrolysis and synthesis of beta-lactam antibiotics. The crystal structures of the native enzyme, both unliganded and in complex with the hydrolysis product D-phenylglycine are reported, as well as the structures of an inactive mutant (S205A) complexed with the substrate ampicillin, and an active site mutant (Y206A) with an increased tendency to catalyze antibiotic production rather than hydrolysis. The structure of the native enzyme shows an acyl binding pocket, in which D-phenylglycine binds, and an additional space that is large enough to accommodate the beta-lactam moiety of an antibiotic. In the S205A mutant, ampicillin binds in this pocket in a non-productive manner, making extensive contacts with the side chain of Tyr(112), which also participates in oxyanion hole formation. In the Y206A mutant, the Tyr(112) side chain has moved with its hydroxyl group toward the catalytic serine. Because this changes the properties of the beta-lactam binding site, this could explain the increased beta-lactam transferase activity of this mutant.     

Catalytic mechanism of glycine N-methyltransferase

Methyltransfer reactions are some of the most important reactions in biological systems. Glycine N-methyltransferase (GNMT) catalyzes the S-adenosyl-l-methionine- (SAM-) dependent methylation of glycine to form sarcosine. Unlike most SAM-dependent methyltransferases, GNMT has a relatively high value and is weakly inhibited by the product S-adenosyl-l-homocysteine (SAH). The major role of GNMT is believed to be the regulation of the cellular SAM/SAH ratio, which is thought to play a key role in SAM-dependent methyltransfer reactions. Crystal structures of GNMT complexed with SAM and acetate (a potent competitive inhibitor of Gly) and the R175K mutated enzyme complexed with SAM were determined at 2.8 and 3.0 A resolutions, respectively. With these crystal structures and the previously determined structures of substrate-free enzyme, a catalytic mechanism has been proposed. Structural changes occur in the transitions from the substrate-free to the binary complex and from the binary to the ternary complex. In the ternary complex stage, an alpha-helix in the N-terminus undergoes a major conformational change. As a result, the bound SAM is firmly connected to protein and a "Gly pocket" is created near the bound SAM. The second substrate Gly binds to Arg175 and is brought into the Gly pocket. Five hydrogen bonds connect the Gly in the proximity of the bound SAM and orient the lone pair orbital on the amino nitrogen (N) of Gly toward the donor methyl group (C(E)) of SAM. Thermal motion of the enzyme leads to a collision of the N and C(E) so that a S(N)2 methyltransfer reaction occurs. The proposed mechanism is supported by mutagenesis studies.     

A mutational analysis of the active site of human type II inosine 5'-monophosphate dehydrogenase

The oxidation of IMP to XMP is the rate-limiting step in the de novo synthesis of guanine ribonucleotides. This NAD-dependent reaction is catalyzed by the enzyme inosine monophosphate dehydrogenase (IMPDH). Based upon the recent structural determination of IMPDH complexed to oxidized IMP (XMP*) and the potent uncompetitive inhibitor mycophenolic acid (MPA), we have selected active site residues and prepared mutants of human type II IMPDH. The catalytic parameters of these mutants were determined. Mutations G326A, D364A, and the active site nucleophile C331A all abolish enzyme activity to less than 0.1% of wild type. These residues line the IMP binding pocket and are necessary for correct positioning of the substrate, Asp364 serving to anchor the ribose ring of the nucleotide. In the MPA/NAD binding site, significant loss of activity was seen by mutation of any residue of the triad Arg322, Asn303, Asp274 which form a hydrogen bonding network lining one side of this pocket. From a model of NAD bound to the active site consistent with the mutational data, we propose that these resides are important in binding the ribose ring of the nicotinamide substrate. Additionally, mutations in the pair Thr333, Gln441, which lies close to the xanthine ring, cause a significant drop in the catalytic activity of IMPDH. It is proposed that these residues serve to deliver the catalytic water molecule required for hydrolysis of the cysteine-bound XMP* intermediate formed after oxidation by NAD.     

Crystal structure of heart 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFKFB2) and the inhibitory influence of citrate on substrate binding

The heart‐specific isoform of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFKFB2) is an important regulator of glycolytic flux in cardiac cells. Here, we present the crystal structures of two PFKFB2 orthologues, human and bovine, at resolutions of 2.0 and 1.8 Å, respectively. Citrate, a TCA cycle intermediate and well‐known inhibitor of PFKFB2, co‐crystallized in the 2‐kinase domains of both orthologues, occupying the fructose‐6‐phosphate binding‐site and extending into the γ‐phosphate binding pocket of ATP. This steric and electrostatic occlusion of the γ‐phosphate site by citrate proved highly consequential to the binding of co‐complexed ATP analogues. The bovine structure, which co‐crystallized with ADP, closely resembled the overall structure of other PFKFB isoforms, with ADP mimicking the catalytic binding mode of ATP. The human structure, on the other hand, co‐complexed with AMPPNP, which, unlike ADP, contains a γ‐phosphate. The presence of this γ‐phosphate made adoption of the catalytic ATP binding mode impossible for AMPPNP, forcing the analogue to bind atypically with concomitant conformational changes to the ATP binding‐pocket. Inhibition kinetics were used to validate the structural observations, confirming citrate's inhibition mechanism as competitive for F6P and noncompetitive for ATP. Together, these structural and kinetic data establish a molecular basis for citrate's negative feed‐back loop of the glycolytic pathway via PFKFB2. Proteins 2016; 85:117–124. © 2016 Wiley Periodicals, Inc.     

Flexibility and enzymatic cold-adaptation: a comparative molecular dynamics investigation of the elastase family

Molecular dynamics simulations of representative mesophilic and psycrophilic elastases have been carried out at different temperatures to explore the molecular basis of cold adaptation inside a specific enzymatic family. The molecular dynamics trajectories have been compared and analyzed in terms of secondary structure, molecular flexibility, intramolecular and protein-solvent interactions, unravelling molecular features relevant to rationalize the efficient catalytic activity of psychrophilic elastases at low temperature. The comparative molecular dynamics investigation reveals that modulation of the number of protein-solvent interactions is not the evolutionary strategy followed by the psycrophilic elastase to enhance catalytic activity at low temperature. In addition, flexibility and solvent accessibility of the residues forming the catalytic triad and the specificity pocket are comparable in the cold- and warm-adapted enzymes. Instead, loop regions with different amino acid composition in the two enzymes, and clustered around the active site or the specificity pocket, are characterized by enhanced flexibility in the cold-adapted enzyme. Remarkably, the psycrophilic elastase is characterized by reduced flexibility, when compared to the mesophilic counterpart, in some scattered regions distant from the functional sites, in agreement with hypothesis suggesting that local rigidity in regions far from functional sites can be beneficial for the catalytic activity of psychrophilic enzymes.     

The crystal structure of phosphinothricin in the active site of glutamine synthetase illuminates the mechanism of enzymatic inhibition

Phosphinothricin is a potent inhibitor of the enzyme glutamine synthetase (GS). The resolution of the native structure of GS from Salmonella typhimurium has been extended to 2.5 A resolution, and the improved model is used to determine the structure of phosphinothricin complexed to GS by difference Fourier methods. The structure suggests a noncovalent, dead-end mechanism of inhibition. Phosphinothricin occupies the glutamate substrate pocket and stabilizes the Glu327 flap in a position which blocks the glutamate entrance to the active site, trapping the inhibitor on the enzyme. One oxygen of the phosphinyl group of phosphinothricin appears to be protonated, because of its proximity to the carboxylate group of Glu327. The other phosphinyl oxygen protrudes into the negatively charged binding pocket for the substrate ammonium, disrupting that pocket. The distribution of charges in the glutamate binding pocket is complementary to those of phosphinothricin. The presence of a second ammonium binding site within the active site is confirmed by its analogue thallous ion, marking the ammonium site and its protein ligands. The inhibition of GS by methionine sulfoximine can be explained by the same mechanism. These models of inhibited GS further illuminate its catalytic mechanism.     

Molecular dynamics simulations of the whey protein beta-lactoglobulin.

Molecular dynamics simulations have been used to model the motions and conformational behavior of the whey protein bovine beta-lactoglobulin. Simulations were performed for the protein by itself and complexed to a single retinol ligand located in a putative interior binding pocket. In the absence of the retinol ligand, the backbone loops around the opening of this interior pocket shifted inward to partially close off this cavity, similar to the shifts observed in previously reported molecular dynamics simulations of the uncomplexed form of the homologous retinol binding protein. The protein complexed with retinol does not exhibit the same conformational shifts. Conformational changes of this type could serve as a recognition signal allowing in vivo discrimination between the free and retinol complexed forms of the beta-lactoglobulin molecule. The unusual bending of the single alpha-helix observed in the simulations of retinol binding protein were not observed in the present calculations.     

Molecular mimicry of substrate oxygen atoms by water molecules in the beta-amylase active site

Soybean beta-amylase (EC 3.2.1.2) has been crystallized both free and complexed with a variety of ligands. Four water molecules in the free-enzyme catalytic cleft form a multihydrogen-bond network with eight strategic residues involved in enzyme-ligand hydrogen bonds. We show here that the positions of these four water molecules are coincident with the positions of four potential oxygen atoms of the ligands within the complex. Some of these waters are displaced from the active site when the ligands bind to the enzyme. How many are displaced depends on the shape of the ligand. This means that when one of the four positions is not occupied by a ligand oxygen atom, the corresponding water remains. We studied the functional/structural role of these four waters and conclude that their presence means that the conformation of the eight side chains is fixed in all situations (free or complexed enzyme) and preserved from unwanted or forbidden conformational changes that could hamper the catalytic mechanism. The water structure at the active pocket of beta-amylase is therefore essential for providing the ligand recognition process with plasticity. It does not affect the protein active-site geometry and preserves the overall hydrogen-bonding network, irrespective of which ligand is bound to the enzyme. We also investigated whether other enzymes showed a similar role for water. Finally, we discuss the potential use of these results for predicting whether water molecules can mimic ligand atoms in the active center.     

The structure of an enzyme-product complex reveals the critical role of a terminal hydroxide nucleophile in the bacterial phosphotriesterase mechanism

A detailed understanding of the catalytic mechanism of enzymes is an important step toward improving their activity for use in biotechnology. In this paper, crystal soaking experiments and X-ray crystallography were used to analyse the mechanism of the Agrobacterium radiobacter phosphotriesterase, OpdA, an enzyme capable of detoxifying a broad range of organophosphate pesticides. The structures of OpdA complexed with ethylene glycol and the product of dimethoate hydrolysis, dimethyl thiophosphate, provide new details of the catalytic mechanism. These structures suggest that the attacking nucleophile is a terminally bound hydroxide, consistent with the catalytic mechanism of other binuclear metallophosphoesterases. In addition, a crystal structure with the potential substrate trimethyl phosphate bound non-productively demonstrates the importance of the active site cavity in orienting the substrate into an approximation of the transition state.     

Analysis of the context dependent sequence requirements of active site residues in the metallo-beta-lactamase IMP-1

The metallo-beta-lactamase IMP-1 catalyzes the hydrolysis of a broad range of beta-lactam antibiotics to provide bacterial resistance to these compounds. In this study, 29 amino acid residue positions in and near the active-site pocket of the IMP-1 enzyme were randomized individually by site-directed mutagenesis of the corresponding codons in the bla(IMP-1) gene. The 29 random libraries were used to identify positions that are critical for the catalytic and substrate-specific properties of the IMP-1 enzyme. Mutants from each of the random libraries were selected for the ability to confer to Escherichia coli resistance to ampicillin, cefotaxime, imipenem or cephaloridine. The DNA sequence of several functional mutants was determined for each of the substrates. Comparison of the sequences of mutants obtained from the different antibiotic selections indicates the sequence requirements for each position in the context of each substrate. The zinc-chelating residues in the active site were found to be essential for hydrolysis of all antibiotics tested. Several positions, however, displayed context-dependent sequence requirements, in that they were essential for one substrate(s) but not others. The most striking examples included Lys69, Asp84, Lys224, Pro225, Gly232, Asn233, Asp236 and Ser262. In addition, comparison of the results for all 29 positions indicates that hydrolysis of imipenem, cephaloridine and ampicillin has stringent sequence requirements, while the requirements for hydrolysis of cefotaxime are more relaxed. This suggests that more information is required to specify active-site pockets that carry out imipenem, cephaloridine or ampicillin hydrolysis than one that catalyzes cefotaxime hydrolysis.     

Insights into the function of the zinc hydroxide-Thr199-Glu106 hydrogen bonding network in carbonic anhydrases

The exact functional role of the zinc hydroxide (water)-Thr199-Glu106 hydrogen bond network in the carbonic anhydrases is unknown. However, from the results of molecular dynamics simulations (MD) we are able to better define its function. From computer graphics analysis and MD simulations on the zinc hydroxide form of human carbonic anhydrase II we find that this interaction forces the hydroxide hydrogen atom to be in a "down" position relative to the deep water-binding pocket. From previous work we have found that this pocket is a high-affinity binding site for CO2. We also note that during the timescale of our simulation (126 ps) the hydrogen bonds between the hydroxide hydrogen atom and Thr199 and the one between Thr199 and Glu106 are not fluxional. We propose that the role of the zinc hydroxide (water)-Thr199-Glu106 hydrogen bond network is to lock the hydrogen atom in the down position in order to expose the CO2 molecule bound in the deep water pocket to a lone pair of the hydroxide oxygen atom. This would allow for the rapid reaction of the CO2 molecule around the zinc ion. Furthermore, if the hydroxide hydrogen atom were not locked in the down position the binding of CO2 to the deep water pocket could be interfered with by the unrestrained hydroxide hydrogen atom (e.g. the N-Zn-O-H torsion could undergo rotational transitions that would partially block the deep water pocket). In summary, the roles we ascribe to this hydrogen bonding network are (1) to allow for facile access of CO2 to the deep water pocket and (2) to allow for maximal exposure of a hydroxide oxygen lone pair to the CO2 carbon atom.     

The structure of an enzyme–product complex reveals the critical role of a terminal hydroxide nucleophile in the bacterial phosphotriesterase mechanism

A detailed understanding of the catalytic mechanism of enzymes is an important step toward improving their activity for use in biotechnology. In this paper, crystal soaking experiments and X-ray crystallography were used to analyse the mechanism of the Agrobacterium radiobacter phosphotriesterase, OpdA, an enzyme capable of detoxifying a broad range of organophosphate pesticides. The structures of OpdA complexed with ethylene glycol and the product of dimethoate hydrolysis, dimethyl thiophosphate, provide new details of the catalytic mechanism. These structures suggest that the attacking nucleophile is a terminally bound hydroxide, consistent with the catalytic mechanism of other binuclear metallophosphoesterases. In addition, a crystal structure with the potential substrate trimethyl phosphate bound non-productively demonstrates the importance of the active site cavity in orienting the substrate into an approximation of the transition state.     

The crystal structure of the ring-hydroxylating dioxygenase from Sphingomonas CHY-1

The ring-hydroxylating dioxygenase (RHD) from Sphingomonas CHY-1 is remarkable due to its ability to initiate the oxidation of a wide range of polycyclic aromatic hydrocarbons (PAHs), including PAHs containing four- and five-fused rings, known pollutants for their toxic nature. Although the terminal oxygenase from CHY-1 exhibits limited sequence similarity with well characterized RHDs from the naphthalene dioxygenase family, the crystal structure determined to 1.85 A by molecular replacement revealed the enzyme to share the same global alpha(3)beta(3) structural pattern. The catalytic domain distinguishes itself from other bacterial non-heme Rieske iron oxygenases by a substantially larger hydrophobic substrate binding pocket, the largest ever reported for this type of enzyme. While residues in the proximal region close to the mononuclear iron atom are conserved, the central region of the catalytic pocket is shaped mainly by the side chains of three amino acids, Phe350, Phe404 and Leu356, which contribute to the rather uniform trapezoidal shape of the pocket. Two flexible loops, LI and LII, exposed to the solvent seem to control the substrate access to the catalytic pocket and control the pocket length. Compared with other naphthalene dioxygenases residues Leu223 and Leu226, on loop LI, are moved towards the solvent, thus elongating the catalytic pocket by at least 2 A. An 11 A long water channel extends from the interface between the alpha and beta subunits to the catalytic site. The comparison of these structures with other known oxygenases suggests that the broad substrate specificity presented by the CHY-1 oxygenase is primarily due to the large size and particular topology of its catalytic pocket and provided the basis for the study of its reaction mechanism.