The crystal structure of mouse phosphoglucose isomerase at 1.6A resolution and its complex with glucose 6-phosphate reveals the catalytic mechanism of sugar ring opening

Phosphoglucose isomerase (PGI) is an enzyme of glycolysis that interconverts glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P) but, outside the cell, is a multifunctional cytokine. High-resolution crystal structures of the enzyme from mouse have been determined in native form and in complex with the inhibitor erythrose 4-phosphate, and with the substrate glucose 6-phosphate. In the substrate-bound structure, the glucose sugar is observed in both straight-chain and ring forms. This structure supports a specific role for Lys518 in enzyme-catalyzed ring opening and we present a "push-pull" mechanism in which His388 breaks the O5-C1 bond by donating a proton to the ring oxygen atom and, simultaneously, Lys518 abstracts a proton from the C1 hydroxyl group. The reverse occurs in ring closure. The transition from ring form to straight-chain substrate is achieved through rotation of the C3-C4 bond, which brings the C1-C2 region into close proximity to Glu357, the base catalyst for the isomerization step. The structure with G6P also explains the specificity of PGI for glucose 6-phosphate over mannose 6-isomerase (M6P). To isomerize M6P to F6P requires a rotation of its C2-C3 bond but in PGI this is sterically blocked by Gln511.     

A new mechanism for the control of phenoloxidase activity: inhibition and complex formation with quinone isomerase

Insect phenoloxidases participate in three physiologically important processes, viz., cuticular hardening (sclerotization), defense reactions (immune reaction), and wound healing. Arrest or even delay of any of these processes compromises the survival of insects. Since the products of phenoloxidase action, viz., quinones, are cytotoxic, uncontrolled phenoloxidase action is deleterious to the insects. Therefore, the activity of this important enzyme has to be finely controlled. A novel inhibition of insect phenoloxidases, which serves as a new regulatory mechanism for control of its activity, is described. The activity of phenoloxidases isolated from both Sarcophaga bullata and Manduca sexta is drastically inhibited by quinone isomerase (isolated from Calliphora), an enzyme that utilizes the phenoloxidase-generated 4-alkylquinones. In turn, phenoloxidase reciprocated the inhibition of isomerase. By forming a complex and controlling each other's activity, these two enzymes seem to regulate the levels of endogenously quinones. In support of this contention, an endogenous complex consisting of phenoloxidase, quinone isomerase, and quinone methide isomerase was characterized from the insect, Calliphora. This sclerotinogenic complex was isolated and purified by borate extraction of the larval cuticle, ammonium sulfate precipitation, and Sepharose 6B column chromatography. The complex exhibited a molecular mass of about 620-680 kDa, as judged by size-exclusion chromatography on Sepharose 6B and HPLC and did not even enter 3% polyacrylamide gel during electrophoresis. The phenoloxidase activity of the complex exhibited a wide substrate specificity. Incubation of the complex with N-acetyldopamine rapidly generated N-acetylnorepinephrine, dehydro-N-acetyldopamine, and its dimers. In addition, transient accumulation of N-acetyldopamine quinone was also observed. These results confirm the presence of phenoloxidase, quinone isomerase, and quinone methide isomerase in the complex. Attempts to dissociate the complex with even trace amounts of SDS ended in the total loss of quinone isomerase activity. The complex does not seems to be made up of stoichiometric amounts of individual enzymes as the ratio of phenoloxidase to quinone isomerase varied from preparation to preparation. It is proposed that the complex formation between sequential enzymes of sclerotinogenic pathway is advantageous for the organism to effectively channel various reactive intermediates during cuticular hardening.     

The structures of the unique sulfotransferase retinol dehydratase with product and inhibitors provide insight into enzyme mechanism and inhibition

The structure of retinol dehydratase (DHR) from Spodoptera frugiperda, a member of the sulfotransferase superfamily, in complexes with the inactive form of the cofactor PAP 3'-phosphoadenosine 5'-phosphate (PAP) and (1) the product of the reaction with retinol anhydroretinol (AR), (2) the retinoid inhibitor all-trans-4-oxoretinol (OR), and (3) the potent steroid inhibitor androsterone (AND) have been determined and compared to the enzyme complex with PAP and retinol. The structures show that the geometry of the active-site amino acids is largely preserved in the various complexes. However, the beta-ionone rings of the retinoids are oriented differently with respect to side chains that have been shown to be important for the enzymatic reaction. In addition, the DHR:PAP:AND complex reveals a novel mode for steroid binding that contrasts significantly with that for steroid binding in other sulfotransferases. The molecule is displaced and rotated approximately 180 degrees along its length so that there is no acceptor hydroxyl in close proximity to the site of sulfate transfer. This observation explains why steroids are potent inhibitors of retinol dehydratase activity, rather than substrates for sulfonation. Most of the steroid-protein contacts are provided by the alpha-helical cap that distinguishes this member of the superfamily. This observation suggests that in addition to providing a chemical environment that promotes the dehydration of a sulfonated intermediate, the cap may also serve to minimize a promiscuous sulfotransferases activity.     

An analysis of structural similarity in the iron and manganese superoxide dismutases based on known structures and sequences

There are two types of homologous enzymes catalysing the dismutationof the superoxide radical – Cu–Zn superoxide dismutases, and manganese oriron superoxide dismutases. In the latter two forms there is a highpercentage of identity in the primary structures, and the tertiarystructures are very similar particu-larly in the areas of the activesite and in the residues responsible for the formation of the dimer.The quaternary structure of the dimer is also highly conserved.However, it has been found that despite this conservation thereis strong metal ion specificity and many enzymes in the familywill only be active if the correct metal ion is present. The purposeof this study has been to analyse solved X-ray structures forinter-actions common in both the manganese and iron forms andthose that are specific to each, which may indi-cate reasonsfor the metal ion specificity. Initial analysis points to theprobability that it is a combination of a number of residues,and not necessarily the same ones in every instance, which conferthe specificity. In addition we have identified some anomalies inthe currently available Fe/MnSOD structures which may require furtherremodelling and refinement. © Rapid Science 1998     

Crystal structures of the T4 phage beta-glucosyltransferase and the D100A mutant in complex with UDP-glucose: glucose binding and identification of the catalytic base for a direct displacement mechanism

T4 phage beta-glucosyltransferase (BGT) is an inverting glycosyltransferase (GT) that transfers glucose from uridine diphospho-glucose (UDP-glucose) to an acceptor modified DNA. BGT belongs to the GT-B structural superfamily, represented, so far, by five different inverting or retaining GT families. Here, we report three high-resolution X-ray structures of BGT and a point mutant solved in the presence of UDP-glucose. The two co-crystal structures of the D100A mutant show that, unlike the wild-type enzyme, this mutation prevents glucose hydrolysis. This strongly indicates that Asp100 is the catalytic base. We obtained the wild-type BGT-UDP-glucose complex by soaking substrate-free BGT crystals. Comparison with a previous structure of BGT solved in the presence of the donor product UDP and an acceptor analogue provides the first model of an inverting GT-B enzyme in which both the donor and acceptor substrates are bound to the active site. The structural analyses support the in-line displacement reaction mechanism previously proposed, locate residues involved in donor substrate specificity and identify the catalytic base.     

Crystal structures of methionine adenosyltransferase complexed with substrates and products reveal the methionine-ATP recognition and give insights into the catalytic mechanism

Methionine adenosyltransferases (MATs) are a family of enzymes in charge of synthesising S-adenosylmethionine (SAM), the most important methyl donor present in living organisms. These enzymes use methionine and ATP as reaction substrates, which react in a S(N)2 fashion where the sulphur atom from methionine attacks C5' from ATP while triphosphate chain is cleaved. A MAT liver specific isoenzyme has been detected, which exists in two distinct oligomeric forms, a dimer (MAT III) and a tetramer (MAT I). Our previously reported crystal structure of MAT I complexed with an inhibitor led to the identification of the methionine-binding site. We present here the results obtained from the complex of MAT I with a competitive inhibitor of methionine, (2S,4S)-amino-4,5-epoxypentanoic acid (AEP), which presents the same features at the methionine binding site reported before. We have also analysed several complexes of this enzyme with methionine and ATP and analogues of them, in order to characterise the interaction that is produced between both substrates. The crystal structures of the complexes reveal how the substrates recognise each other at the active site of the enzyme, and suggest a putative binding site for the product SAM. The residues involved in the interactions of substrates and products with MAT have been identified, and the results agree with all the previous data concerning mutagenesis experiments and crystallographic work. Moreover, all the information provided from the analysis of the complexes has allowed us to postulate a catalytic mechanism for this family of enzymes. In particular, we propose a key role for Lys182 in the correct positioning of the substrates, and Asp135(*), in stabilising the sulphonium group formed in the product (SAM).     

A nanogold resonance Rayleigh scattering method for determination of trace As based on the hydride nanoreaction

In H₂SO₄ solution, As(III) was reduced to arsine (AsH₃) by NaBH₄, and was absorbed in HAuCl₄ solution to form nanogold particles (NGs) that exhibited a resonance Rayleigh scattering (RRS) effect at 370 nm. Under the selected conditions, when the As(III) concentration increased the RRS peak also increased due to the formation of more NGs. There was a linear correlation between RRS intensity and As(III) concentration in the range 6–1000 ng/mL, with a detection limit of 3 ng/mL. This new hydride generation–nanogold reaction RRS (HG–NG RRS) method was applied to determine trace amounts of As in milk samples, with satisfactory results. Copyright © 2015 John Wiley & Sons, Ltd.     

Crystal structures of Tritrichomonasfoetus inosine monophosphate dehydrogenase in complex with substrate,cofactor and analogs: a structural basis for the random-in ordered-out kinetic mechanism

The enzyme inosine monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide biosynthesis. Because it is up-regulated in rapidly proliferating cells, human type II IMPDH is actively targeted for immunosuppressive, anticancer, and antiviral chemotherapy. The enzyme employs a random-in ordered-out kinetic mechanism where substrate or cofactor can bind first but product is only released after the cofactor leaves. Due to structural and kinetic differences between mammalian and microbial enzymes, most drugs that are successful in the inhibition of mammalian IMPDH are far less effective against the microbial forms of the enzyme. It is possible that with greater knowledge of the structural mechanism of the microbial enzymes, an effective and selective inhibitor of microbial IMPDH will be developed for use as a drug against multi-drug resistant bacteria and protists. The high-resolution crystal structures of four different complexes of IMPDH from the protozoan parasite Tritrichomonas foetus have been solved: with its substrate IMP, IMP and the inhibitor mycophenolic acid (MPA), the product XMP with MPA, and XMP with the cofactor NAD(+). In addition, a potassium ion has been located at the dimer interface. A structural model for the kinetic mechanism is proposed.     

Crystal structures of Vibrio harveyi chitinase A complexed with chitooligosaccharides: implications for the catalytic mechanism

This research describes four X-ray structures of Vibrio harveyi chitinase A and its catalytically inactive mutant (E315M) in the presence and absence of substrates. The overall structure of chitinase A is that of a typical family-18 glycosyl hydrolase comprising three distinct domains: (i) the amino-terminal chitin-binding domain; (ii) the main catalytic (α/β)₈ TIM-barrel domain; and (iii) the small (α + β) insertion domain. The catalytic cleft of chitinase A has a long, deep groove, which contains six chitooligosaccharide ring-binding subsites (−4)(−3)(−2)(−1)(+1)(+2). The binding cleft of the ligand-free E315M is partially blocked by the C-terminal (His)₆-tag. Structures of E315M-chitooligosaccharide complexes display a linear conformation of pentaNAG, but a bent conformation of hexaNAG. Analysis of the final 2F_o − F_c omit map of E315M-NAG6 reveals the existence of the linear conformation of the hexaNAG at a lower occupancy with respect to the bent conformation. These crystallographic data provide evidence that the interacting sugars undergo conformational changes prior to hydrolysis by the wild-type enzyme.     

The solution structure of eglin c based on measurements of many NOEs and coupling constants and its comparison with X-ray structures.

A high-precision solution structure of the elastase inhibitor eglin c was determined by NMR and distance geometry calculations. A large set of 947 nuclear Overhauser (NOE) distance constraints was identified, 417 of which were quantified from two-dimensional NOE spectra at short mixing times. In addition, a large number of homonuclear 1H-1H and heteronuclear 1H-15N vicinal coupling constants were used, and constraints on 42 chi 1 and 38 phi angles were obtained. Structure calculations were carried out using the distance geometry program DG-II. These calculations had a high convergence rate, in that 66 out of 75 calculations converged with maximum residual NOE violations ranging from 0.17 A to 0.47 A. The spread of the structures was characterized with average root mean square deviations () between the structures and a mean structure. To calculate the unbiased toward any single structure, a new procedure was used for structure alignment. A canonical structure was calculated from the mean distances, and all structures were aligned relative to that. Furthermore, an angular order parameter S was defined and used to characterize the spread of structures in torsion angle space. To obtain an accurate estimate of the precision of the structure, the number of calculations was increased until the and the angular order parameters stabilized. This was achieved after approximately 40 calculations. The structure consists of a well-defined core whose backbone deviates from the canonical structure ca. 0.4 A, a disordered N-terminal heptapeptide whose backbone deviates by 0.8-12 A, and a proteinase-binding loop whose backbone deviates up to 3.0 A. Analysis of the angular order parameters and inspection of the structures indicates that a hinge-bending motion of the binding loop may occur in solution. Secondary structures were analyzed by comparison of dihedral angle patterns. The high precision of the structure allows one to identify subtle differences with four crystal structures of eglin c determined in complexes with proteinases.     

A base-flipping mechanism for the T4 phage beta-glucosyltransferase and identification of a transition-state analog

T4 phage beta-glucosyltransferase (BGT) modifies T4 DNA. We crystallized BGT with UDP-glucose and a 13mer DNA fragment containing an abasic site. We obtained two crystal structures of a ternary complex BGT-UDP-DNA at 1.8A and 2.5A resolution, one with a Tris molecule and the other with a metal ion at the active site. Both structures reveal a large distortion in the bound DNA. BGT flips the deoxyribose moiety at the abasic site to an extra-helical position and induces a 40 degrees bend in the DNA with a marked widening of the major groove. The Tris molecule mimics the glucose moiety in its transition state. The base-flipping mechanism, which has so far been observed only for glycosylases, methyltransferases and endonucleases, is now reported for a glucosyltransferase. BGT is unique in binding and inserting a loop into the DNA duplex through the major groove only. Furthermore, BGT compresses the backbone DNA one base further than the target base on the 3'-side.     

A general modeling framework for open wildlife populations based on the Polya tree prior

Wildlife monitoring for open populations can be performed using a number of different survey methods. Each survey method gives rise to a type of data and, in the last five decades, a large number of associated statistical models have been developed for analyzing these data. Although these models have been parameterized and fitted using different approaches, they have all been designed to either model the pattern with which individuals enter and/or exit the population, or to estimate the population size by accounting for the corresponding observation process, or both. However, existing approaches rely on a predefined model structure and complexity, either by assuming that parameters linked to the entry and exit pattern (EEP) are specific to sampling occasions, or by employing parametric curves to describe the EEP. Instead, we propose a novel Bayesian nonparametric framework for modeling EEPs based on the Polya tree (PT) prior for densities. Our Bayesian nonparametric approach avoids overfitting when inferring EEPs, while simultaneously allowing more flexibility than is possible using parametric curves. Finally, we introduce the replicate PT prior for defining classes of models for these data allowing us to impose constraints on the EEPs, when required. We demonstrate our new approach using capture–recapture, count, and ring-recovery data for two different case studies.     

A metallo-beta-lactamase enzyme in action: crystal structures of the monozinc carbapenemase CphA and its complex with biapenem

One strategy developed by bacteria to resist the action of beta-lactam antibiotics is the expression of metallo-beta-lactamases. CphA from Aeromonas hydrophila is a member of a clinically important subclass of metallo-beta-lactamases that have only one zinc ion in their active site and for which no structure is available. The crystal structures of wild-type CphA and its N220G mutant show the structural features of the active site of this enzyme, which is modeled specifically for carbapenem hydrolysis. The structure of CphA after reaction with a carbapenem substrate, biapenem, reveals that the enzyme traps a reaction intermediate in the active site. These three X-ray structures have allowed us to propose how the enzyme recognizes carbapenems and suggest a mechanistic pathway for hydrolysis of the beta-lactam. This will be relevant for the design of metallo-beta-lactamase inhibitors as well as of antibiotics that escape their hydrolytic activity.     

A sensitive fluorescent sensor based on the photoinduced electron transfer mechanism for cefixime and ctDNA

Cefixime is a third generation orally administered cephalosporin that is frequently used as a broad spectrum antibiotic against various gram‐negative and gram‐positive bacteria. In this study, a simple and sensitive fluorescent sensor for the determination of the cefixime and ctDNA was established based on the CdTe:Zn²⁺ quantum dots (QDs). The fluorescence of CdTe:Zn²⁺ QDs can be effectively quenched by cefixime in virtue of the surface binding of cefixime on CdTe:Zn²⁺ QDs and the subsequent photoinduced electron transfer process from CdTe:Zn²⁺ QDs to cefixime, in particular, the high sensitivity of QDs fluorescence emission to cefixime at the micromole per liter level, which render the cefixime‐CdTe:Zn²⁺ QDs system into fluorescence “OFF” status, then turn on in the presence of ctDNA. Furthermore, the Fourier transform infrared (FTIR) spectra of characteristic bands of C–N and N–H groups of cefixime endow evidence for the interaction of cefixime with CdTe:Zn²⁺ QDs. The relative electrochemical behavior of the affinity of CdTe:Zn²⁺ QDs for cefixime and ctDNA reveals the potential molecular binding mechanism.     

The crystal structures of oxidized forms of human peroxiredoxin 5 with an intramolecular disulfide bond confirm the proposed enzymatic mechanism for atypical 2-Cys peroxiredoxins

Peroxiredoxin 5 (PRDX5) belongs to the PRDX superfamily of thiol-dependent peroxidases able to reduce hydrogen peroxide, alkyl hydroperoxides and peroxynitrite. PRDX5 is classified in the atypical 2-Cys subfamily of PRDXs. In this subfamily, the oxidized form of the enzyme is characterized by the presence of an intramolecular disulfide bridge between the peroxidatic and the resolving cysteine residues. We report here three crystal forms in which this intramolecular disulfide bond is indeed observed. The structures are characterized by the expected local unfolding of the peroxidatic loop, but also by the unfolding of the resolving loop. A new type of interface between PRDX molecules is described. The three crystal forms were not oxidized in the same way and the influence of the oxidizing conditions is discussed.     

A comparison of the predicted and X-ray structures of angiogenin. Implications for further studies of model building of homologous proteins

The three-dimensional structure of human angiogenin has been determined by X-ray crystallography and is compared here with an earlier model which predicted its structure, based on the homology of angiogenin with bovine pancreatic ribonuclease A. Comparison of the predicted model and crystal structure shows that the active-site histidine residues and the core of the angiogenin molecule, including most of the-strands and-helices, were predicted reasonably well. However, the structure of the surface loop regions and residues near the truncated C-terminus differs significantly. The C-terminal segment includes the active-site residues Asp-116, Gln-117, and Ser-118; Gln-117 in particular has been shown to be important in affecting the ribonucleolytic activity of angiogenin. Also, the orientation of one helix in the model differed from the orientation observed experimentally by about 20°, resulting in a large displacement of this chain segment. The difficulty encountered in predicting the surface loop regions has led to a new algorithm [Palmer and Scheraga (1991),J. Comput. Chem.,12, 505–526; (1992),J. Comput. Chem.,13, 329–350] for predicting the conformations of surface loops.     

A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels.

BACKGROUND: Urease catalyzes the hydrolysis of urea, the final step of organic nitrogen mineralization, using a bimetallic nickel centre. The role of the active site metal ions and amino acid residues has not been elucidated to date. Many pathologies are associated with the activity of ureolytic bacteria, and the efficiency of soil nitrogen fertilization with urea is severely decreased by urease activity. Therefore, the development of urease inhibitors would lead to a reduction of environmental pollution, to enhanced efficiency of nitrogen uptake by plants, and to improved therapeutic strategies for treatment of infections due to ureolytic bacteria. Structure-based design of urease inhibitors would require knowledge of the enzyme mechanism at the molecular level. RESULTS: The structures of native and inhibited urease from Bacillus pasteurii have been determined at a resolution of 2.0 A by synchrotron X-ray cryogenic crystallography. In the native enzyme, the coordination sphere of each of the two nickel ions is completed by a water molecule and a bridging hydroxide. A fourth water molecule completes a tetrahedral cluster of solvent molecules. The enzyme crystallized in the presence of phenylphosphorodiamidate contains the tetrahedral transition-state analogue diamidophosphoric acid, bound to the two nickel ions in an unprecedented mode. Comparison of the native and inhibited structures reveals two distinct conformations of the flap lining the active-site cavity. CONCLUSIONS: The mode of binding of the inhibitor, and a comparison between the native and inhibited urease structures, indicate a novel mechanism for enzymatic urea hydrolysis which reconciles the available structural and biochemical data.