Honggao Yan

Michigan State University, East Lansing, Michigan, United States

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Publications (43)173.04 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Two valid targets for antibiotic development, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS), catalyze consecutive reactions in folate biosynthesis. In Francisella tularensis (Ft), these two activities are contained in a single protein, FtHPPK-DHPS. While Pemble and coworkers determined the structure of FtHPPK-DHPS, they were unable to measure the kinetic parameters of the enzyme (PloS one 5, e14165). In this study, we elucidated the binding and inhibitory activities of two HPPK inhibitors (HP-18 and HP-26) against FtHPPK-DHPS, determined the structure of FtHPPK-DHPS in complex with HP-26, and measured the kinetic parameters for the dual enzymatic activities of FtHPPK-DHPS. The biochemical analyses showed that HP-18 and HP-26 have significant isozyme selectivity and that FtHPPK-DHPS is unique in that the catalytic efficiency of its DHPS activity is only 1/2.6×10(5) that of Escherichia coli DHPS. Sequence and structural analyses suggest that HP-26 is an excellent lead for developing tularemia therapeutics and that the very low DHPS activity is due, at least in part, to the lack of a key residue that interacts with the substrate p-aminobenzoic acid (pABA). A BLAST search of 10 F. tularensis genomes indicated that the bacterium contains a single FtHPPK-DHPS. The marginal DHPS activity and the singular existence of FtHPPK-DHPS in F. tularensis make this bacterium more vulnerable to DHPS inhibitors. Current sulfa drugs are ineffective against tularemia; new inhibitors targeting the unique pABA-binding pocket may be effective and less subject to resistance because mutation may make the marginal DHPS activity unable to support the growth of F. tularensis. This article is protected by copyright. All rights reserved.
    FEBS Journal 06/2014; · 4.25 Impact Factor
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    ABSTRACT: SpoIIID is evolutionarily conserved in endospore-forming bacteria and it activates or represses many genes during sporulation of Bacillus subtilis. A SpoIIID monomer binds DNA with high affinity and moderate sequence specificity. In addition to a predicted helix-turn-helix motif, SpoIIID has a C-terminal basic region that contributes to DNA binding. The NMR solution structure of SpoIIID in complex with DNA revealed that SpoIIID does indeed have a helix-turn-helix domain and that it has a novel C-terminal helical extension. Residues in both these regions interact with DNA, based on the NMR data and on the effects on DNA binding in vitro of SpoIIID with single-alanine substitutions. These data, as well as sequence conservation in SpoIIID binding sites, were used for information-driven docking to model the SpoIIID⋅DNA complex. The modeling resulted in a single cluster of models in which the recognition helix of the helix-turn-helix domain interacts with the major groove of DNA, as expected. Interestingly, the C-terminal extension, which includes two helices connected by a kink, interacts with the adjacent minor groove of DNA in the models. This predicted novel mode of binding is proposed to explain how a monomer of SpoIIID achieves high-affinity DNA binding. Since SpoIIID is conserved only in endospore-forming bacteria, which include important pathogenic Bacilli and Clostridia whose ability to sporulate contributes to their environmental persistence, the interaction of the C-terminal extension of SpoIIID with DNA is a potential target for development of sporulation inhibitors.
    Journal of bacteriology 02/2014; · 3.94 Impact Factor
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    ABSTRACT: 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthesis pathway catalyzing the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin, is an attractive target for developing novel antimicrobial agents. Previously, we studied the mechanism of HPPK action, synthesized bisubstrate analog inhibitors by linking 6-hydroxymethylpterin to adenosine through phosphate groups, and developed a new generation of bisubstrate inhibitors by replacing the phosphate bridge with a piperidine-containing linkage. To further improve linker properties, we have synthesized a new compound, characterized its protein binding/inhibiting properties, and determined its structure in complex with HPPK. Surprisingly, this inhibitor exhibits a new binding mode in that the adenine base is flipped when compared to previously reported structures. Furthermore, the side chain of amino acid residue E77 is involved in protein-inhibitor interaction, forming hydrogen bonds with both 2' and 3' hydroxyl groups of the ribose moiety. Residue E77 is conserved among HPPK sequences, but interacts only indirectly with the bound MgATP via water molecules. Never observed before, the E77-ribose interaction is compatible only with the new inhibitor-binding mode. Therefore, this compound represents a new direction for further development.
    Bioorganic & medicinal chemistry 06/2012; 20(14):4303-9. · 2.82 Impact Factor
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    ABSTRACT: Yeast cytosine deaminase (yCD) catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, nuclear magnetic resonance (NMR), and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in k(cat) and a dramatic increase in K(m), indicating Glu64 is important for both binding and catalysis in the activation of 5FC. (19)F NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild-type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. (1)H and (15)N NMR analysis revealed trans-hydrogen bond D/H isotope effects on the hydrogen of the amide of Glu64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild-type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has a higher activation energy, as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by (1)H and (15)N NMR analysis. To explore the functional role of Glu64 in catalysis, we investigated the deamination of cytosine catalyzed by the E64A mutant by ONIOM calculations. The results showed that without the assistance of Glu64, both proton transfers before and after the formation of the tetrahedral reaction intermediate become partially rate-limiting steps. The results of the experimental and computational studies together indicate that Glu64 plays a critical role in both the binding and the chemical transformation in the conversion of the prodrug 5FC to the anticancer drug 5-fluorouracil.
    Biochemistry 12/2011; 51(1):475-86. · 3.38 Impact Factor
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    ABSTRACT: 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthetic pathway, catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin. The enzyme is essential for microorganisms, is absent from humans, and is not the target for any existing antibiotics. Therefore, HPPK is an attractive target for developing novel antimicrobial agents. Previously, we characterized the reaction trajectory of HPPK-catalyzed pyrophosphoryl transfer and synthesized a series of bisubstrate analog inhibitors of the enzyme by linking 6-hydroxymethylpterin to adenosine through 2, 3, or 4 phosphate groups. Here, we report a new generation of bisubstrate analog inhibitors. To improve protein binding and linker properties of such inhibitors, we have replaced the pterin moiety with 7,7-dimethyl-7,8-dihydropterin and the phosphate bridge with a piperidine linked thioether. We have synthesized the new inhibitors, measured their K(d) and IC(50) values, determined their crystal structures in complex with HPPK, and established their structure-activity relationship. 6-Carboxylic acid ethyl ester-7,7-dimethyl-7,8-dihydropterin, a novel intermediate that we developed recently for easy derivatization at position 6 of 7,7-dimethyl-7,8-dihydropterin, offers a much high yield for the synthesis of bisubstrate analogs than that of previously established procedure.
    Bioorganic & medicinal chemistry 11/2011; 20(1):47-57. · 2.82 Impact Factor
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    ABSTRACT: The relationship between protein conformational dynamics and enzymatic reactions has been a fundamental focus in modern enzymology. Using single-molecule fluorescence resonance energy transfer (FRET) with a combined statistical data analysis approach, we have identified the intermittently appearing coherence of the enzymatic conformational state from the recorded single-molecule intensity-time trajectories of enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in catalytic reaction. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multistep conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. The coherence frequency, identified by statistical results of the correlation function analysis from single-molecule FRET trajectories, increases with the increasing substrate concentrations. The intermittent coherence in conformational state changes at the enzymatic reaction active site is likely to be common and exist in other conformation regulated enzymatic reactions. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.
    Journal of the American Chemical Society 08/2011; 133(36):14389-95. · 10.68 Impact Factor
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    Honggao Yan, Xinhua Ji
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    ABSTRACT: Enzymatic catalysis has conflicting structural requirements of the enzyme. In order for the enzyme to form a Michaelis complex, the enzyme must be in an open conformation so that the substrate can get into its active center. On the other hand, in order to maximize the stabilization of the transition state of the enzymatic reaction, the enzyme must be in a closed conformation to maximize its interactions with the transition state. The conflicting structural requirements can be resolved by a flexible active center that can sample both open and closed conformational states. For a bisubstrate enzyme, the Michaelis complex consists of two substrates in addition to the enzyme. The enzyme must remain flexible upon the binding of the first substrate so that the second substrate can get into the active center. The active center is fully assembled and stabilized only when both substrates bind to the enzyme. However, the side-chain positions of the catalytic residues in the Michaelis complex are still not optimally aligned for the stabilization of the transition state, which lasts only approximately 10(-13) s. The instantaneous and optimal alignment of catalytic groups for the transition state stabilization requires a dynamic enzyme, not an enzyme which undergoes a large scale of movements but an enzyme which permits at least a small scale of adjustment of catalytic group positions. This review will summarize the structure, catalytic mechanism, and dynamic properties of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase and examine the role of protein conformational dynamics in the catalysis of a bisubstrate enzymatic reaction.
    Protein and Peptide Letters 01/2011; 18(4):328-35. · 1.99 Impact Factor
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    ABSTRACT: Backbone conformational dynamics of Thermotoga neapolitana adenylate kinase in the free form (TNAK) and inhibitor-bound form (TNAK*Ap5A) were investigated at 30 degrees C using (15)N NMR relaxation measurements and NMR monitored hydrogen-deuterium exchange. With kinetic parameters identical to those of Escherichia coli AK (ECAK) at 30 degrees C, TNAK is a unique hyperthermophilic enzyme. These catalytic properties make TNAK an interesting and novel model to study the interplay between protein rigidity, stability, and activity. Comparison of fast time scale dynamics (picosecond to nanosecond) in the open and closed states of TNAK and ECAK at 30 degrees C reveals a uniformly higher rigidity across all domains of TNAK. Within this framework of a rigid TNAK structure, several residues located in the AMP-binding domain and in the core-lid hinge regions display high picosecond to nanosecond time scale flexibility. Together with the recent comparison of ECAK dynamics with those of hyperthermophilic Aquifex aeolicus AK (AAAK), our results provide strong evidence for the role of picosecond to nanosecond time scale fluctuations in both stability and activity. In the slow time scales, TNAK's increased rigidity is not uniform but localized in the AMP-binding and lid domains. The core domain amides of ECAK and TNAK in the open and closed states show comparable protection against exchange. Significantly, the hinges framing the lid domain show similar exchange data in ECAK and TNAK open and closed forms. Our NMR relaxation and hydrogen-deuterium exchange studies therefore suggest that TNAK maintains high activity at 30 degrees C by localizing flexibility to the hinge regions that are key to facilitating conformational changes.
    Biochemistry 03/2009; 48(12):2723-39. · 3.38 Impact Factor
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    ABSTRACT: 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), which follows an ordered bi-bi kinetic mechanism with ATP binding to the enzyme first. HPPK undergoes dramatic conformational changes during its catalytic cycle as revealed by X-ray crystallography, and the conformational changes are essential for the enzymatic catalysis as shown by site-directed mutagenesis and biochemical and crystallographic analysis of the mutants. However, the dynamic properties of the enzyme have not been measured experimentally. Here, we report a (15)N NMR relaxation study of the dynamic properties of Escherichia coli HPPK from the apo form to the binary substrate complex with MgATP (represented by MgAMPCPP, an ATP analogue) to the Michaelis complex (ternary substrate complex) with MgATP (represented by MgAMPCPP) and HP (represented by 7,7-dimethyl-6-hydroxypterin, an HP analogue). The results show that the binding of the nucleotide to HPPK does not cause major changes in the dynamic properties of the enzyme. Whereas enzymes are often more rigid when bound to the ligand or the substrate, the internal mobility of HPPK is not reduced and is even moderately increased in the binary complex, particularly in the catalytic loops. The internal mobility of the catalytic loops is significantly quenched upon the formation of the ternary complex, but some mobility remains. The enhanced motions in the catalytic loops of the binary substrate complex may be required for the assembling of the ternary complex. On the other hand, some degrees of mobility in the catalytic loops of the ternary complex may be required for the optimal stabilization of the transition state, which may need the instantaneous adjustment and alignment of the side-chain positions of catalytic residues. Such dynamic behaviors may be characteristic of bisubstrate enzymes.
    Biochemistry 01/2009; 48(2):302-12. · 3.38 Impact Factor
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    ABSTRACT: The configuration and hydrogen-bonding network of side-chain amides in a 35 kDa protein were determined by measuring differential and trans-hydrogen-bond H/D isotope effects by using the isotopomer-selective (IS)-TROSY technique, which leads to a reliable recognition and correction of erroneous rotamers that are frequently found in protein structures. First, the differential two-bond isotope effects on carbonyl (13)C' shifts, which are defined as Delta(2)Delta(13)C'(ND) = (2)Delta(13)C'(ND(E))-(2)Delta(13)C'(ND(Z)), provide a reliable means for the configuration assignment for side-chain amides, because environmental effects (hydrogen bonds and charges, etc.) are greatly attenuated over the two bonds that separate the carbon and hydrogen atoms, and the isotope effects fall into a narrow range of positive values. Second and more importantly, the significant variations in the differential one-bond isotope effects on (15)N chemical shifts, which are defined as Delta(1)Delta(15)N(D) = (1)Delta(15)N(D(E))-(1)Delta(15)N(D(Z)) can be correlated with hydrogen-bonding interactions, particularly those involving charged acceptors. The differential one-bond isotope effects are additive, with major contributions from intrinsic differential conjugative interactions between the E and Z configurations, H-bonding interactions, and charge effects. Furthermore, the pattern of trans-H-bond H/D isotope effects can be mapped onto more complicated hydrogen-bonding networks that involve bifurcated hydrogen-bonds. Third, the correlations between Delta(1)Delta(15)N(D) and hydrogen-bonding interactions afford an effective means for the correction of erroneous rotamer assignments of side-chain amides. Rotamer correction by differential isotope effects is not only robust, but also simple and can be applied to large proteins.
    ChemBioChem 12/2008; 9(17):2860-71. · 3.74 Impact Factor
  • Journal of the American Chemical Society 03/2008; 130(8):2428-9. · 10.68 Impact Factor
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    ABSTRACT: 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate-biosynthetic pathway and is essential for microorganisms but absent from mammals. HPPK catalyzes Mg(2+)-dependent pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). Previously, three-dimensional structures of Escherichia coli HPPK (EcHPPK) have been determined at almost every stage of its catalytic cycle and the reaction mechanism has been established. Here, the crystal structure of Yersinia pestis HPPK (YpHPPK) in complex with HP and an ATP analog is presented together with thermodynamic and kinetic characterizations. The two HPPK molecules differ significantly in a helix-loop area (alpha2-Lp3). YpHPPK has lower affinities than EcHPPK for both nucleotides and HP, but its rate constants for the mechanistic steps of both chemical transformation and product release are comparable with those of EcHPPK. Y. pestis, which causes plague, is a category A select agent according to the Centers for Disease Control and Prevention (CDC). Therefore, these structural and biochemical data are valuable for the design of novel medical countermeasures against plague.
    Acta Crystallographica Section D Biological Crystallography 12/2007; 63(Pt 11):1169-77. · 14.10 Impact Factor
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    ABSTRACT: The shikimate biosynthetic pathway is essential to microorganisms, plants, and parasites but absent from mammals. Therefore, shikimate dehydrogenase (SD) and other enzymes in the pathway are attractive targets for developing nontoxic antimicrobial agents, herbicides, and antiparasite drugs. SD catalyzes the fourth reaction in the pathway, the nicotinamide adenine dinucleotide phosphate- (NADP-) dependent reduction of 3-dehydroshikimic acid to shikimic acid (SA), as well as its reverse, by the transfer of a hydride. Previous structural studies reveal that the enzyme exists in two major conformations, an open and a closed form. For the reaction to occur, it is believed that the catalytic complex assumes the closed conformation. Nonetheless, the only structure containing both SA and NADP+ exhibits an open conformation (PDB entry 2EV9). Here, we present two crystal structures of Aquifex aeolicus SD, including a ternary complex with both SA and NADP+, which assumes the closed conformation and therefore contains a catalytically competent active site. On the basis of preexisting and novel structural and biochemical data, a catalytic mechanism is proposed.
    Biochemistry 09/2007; 46(33):9513-22. · 3.38 Impact Factor
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    Aizhuo Liu, Lishan Yao, Yue Li, Honggao Yan
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    ABSTRACT: By using the mixed solvent of 50% H2O/50% D2O and employing deuterium decoupling, TROSY experiments exclusively detect NMR signals from semideuterated isotopomers of carboxamide groups with high sensitivities for proteins with molecular weights up to 80 kDa. This isotopomer-selective strategy extends TROSY experiments from exclusively detecting backbone to both backbone and side-chain amides, particularly in large proteins. Because of differences in both TROSY effect and dynamics between 15N-H(E){D(Z)} and 15N-H(Z){D(E)} isotopomers of the same carboxamide, the 15N transverse magnetization of the latter relaxes significantly faster than that of the former, which provides a direct and reliable stereospecific distinction between the two configurations. The TROSY effects on the 15N-H(E){D(Z)} isotopomers of side-chain amides are as significant as on backbone amides.
    Journal of Magnetic Resonance 07/2007; 186(2):319-26. · 2.30 Impact Factor
  • Yi Wang, Yue Li, Yan Wu, Honggao Yan
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    ABSTRACT: Dihydroneopterin aldolase (DHNA) catalyzes both the cleavage of 7,8-dihydro-D-neopterin (DHNP) to form 6-hydroxymethyl-7,8-dihydropterin (HP) and glycolaldehyde and the epimerization of DHNP to form 7,8-dihydro-L-monapterin (DHMP). Whether the epimerization reaction uses the same reaction intermediate as the aldol reaction or the deprotonation and reprotonation of C2' of DHNP has been investigated by NMR analysis of the reaction products in a D2O solvent. No deuteration of C2' was observed for the newly formed DHMP. This result strongly suggests that the epimerization reaction uses the same reaction intermediate as the aldol reaction. In contrast with an earlier observation, the DHNA-catalyzed reaction is reversible, which also supports a nonstereospecific retroaldol/aldol mechanism for the epimerization reaction. The binding and catalytic properties of DHNAs from both Staphylococcus aureus (SaDHNA) and Escherichia coli (EcDHNA) were determined by equilibrium binding and transient kinetic studies. A complete set of kinetic constants for both the aldol and epimerization reactions according to a unified kinetic mechanism was determined for both SaDHNA and EcDHNA. The results show that the two enzymes have significantly different binding and catalytic properties, in accordance with the significant sequence differences between them.
    FEBS Journal 06/2007; 274(9):2240-52. · 4.25 Impact Factor
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    ABSTRACT: Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and the epimerization of DHNP to 7,8-dihydromonopterin (DHMP). Although crystal structures of the enzyme from several microorganisms have been reported, no structural information is available about the critical interactions between DHNA and the trihydroxypropyl moiety of the substrate, which undergoes bond cleavage and formation. Here, we present the structures of Staphylococcus aureus DHNA (SaDHNA) in complex with neopterin (NP, an analog of DHNP) and with monapterin (MP, an analog of DHMP), filling the gap in the structural analysis of the enzyme. In combination with previously reported SaDHNA structures in its ligand-free form (PDB entry 1DHN) and in complex with HP (PDB entry 2DHN), four snapshots for the catalytic center assembly along the reaction pathway can be derived, advancing our knowledge about the molecular mechanism of SaDHNA-catalyzed reactions. An additional step appears to be necessary for the epimerization of DHMP to DHNP. Three active site residues (E22, K100, and Y54) function coordinately during catalysis: together, they organize the catalytic center assembly, and individually, each plays a central role at different stages of the catalytic cycle.
    Journal of Molecular Biology 05/2007; 368(1):161-9. · 3.91 Impact Factor
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    Lishan Yao, Honggao Yan, Robert I Cukier
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    ABSTRACT: Yeast cytosine deaminase, a zinc metalloenzyme, catalyzes the deamination of cytosine to uracil. Experimental and computational evidence indicates that the rate-limiting step is product release, instead of the chemical reaction step. In this work, we use molecular dynamics to suggest ligand exit paths. Simulation at 300 K shows that the active site is well protected by the C-terminal helix (residues 150-158) and F-114 loop (residues 111-117) and that on the molecular dynamics timescale water does not flow in or out of the active site. In contrast, simulation at 320 K shows a significant increase in flexibility of the C-terminal helix and F-114 loop. The motions of these two regions at 320 K open the active site and permit water molecules to diffuse into and out of the active site through two paths with one much more favored than the other. Cytosine is pushed out of the active site by a restraint method in two directions specified by these two paths. In path 1 the required motion of the protein is local-involving only the C-terminal helix and F-114 loop-and two residues, F-114 and I-156, are identified that have to be moved away to let cytosine out; whereas in path 2, the protein has to rearrange itself much more extensively, and the changes are also much larger compared to the path 1 simulation.
    Biophysical Journal 05/2007; 92(7):2301-10. · 3.67 Impact Factor
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    Lishan Yao, Robert I Cukier, Honggao Yan
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    ABSTRACT: The catalytic mechanism of Bacillus subtilis guanine deaminase (bGD), a Zn metalloenzyme, has been investigated by a combination of quantum mechanical calculations using the multilayered ONIOM method and molecular dynamics simulations. In contrast to a previously proposed catalytic mechanism, which requires the bound guanine to assume a rare tautomeric state, the ONIOM calculations showed that the active-site residues of the enzyme do not affect the tautomeric state of guanine, and consequently the bound guanine is a tautomer that is the most abundant in aqueous solution. Two residues, Glutamate 55 and Aspartate 114, were found to play important roles in proton shuttling in the reaction. The proposed reaction path is initiated by proton transfer from a Zn-bound water to protonate Asp114. This process may be quite complex and rather dynamic in nature, as revealed by the molecular dynamics (MD) simulations, whereby another water may bridge the Zn-bound water and Asp114, which then is eliminated by positioning of guanine in the active site. The binding of guanine stabilizes protonated Asp114 by hydrogen bond formation. Asp114 can then transfer its proton to the N3 of the bound guanine, facilitating the nucleophilic attack on C2 of the guanine by the Zn-bound hydroxide to form a tetrahedral intermediate. This occurs with a rather low barrier. Glu55 then transfers a proton from the Zn-hydroxide to the amino group of the reaction intermediate and, at this point, the C2-N2 bond has lengthened by 0.2 A compared to guanine, making C2-N2 bond cleavage more facile. The C2-N2 bond breaks forming ammonia, with an energy barrier of approximately 8.8 kcal/mol. Ammonia leaves the active site, and xanthine is freed by the cleavage of the Zn-O2 bond, with a barrier approximately 8.4 kcal/mol. Along this reaction path, the highest barrier comes from C2-N2 bond cleavage, while the barrier from the cleavage of the Zn-O2 bond is slightly smaller. The Zn-O2 bond can be broken without the assistance of water during the release of xanthine.
    The Journal of Physical Chemistry B 04/2007; 111(16):4200-10. · 3.61 Impact Factor
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    Aizhuo Liu, Yue Li, Lishan Yao, Honggao Yan
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    ABSTRACT: A new strategy for the simultaneous NMR assignment of both backbone and side chain amides in large proteins with isotopomer-selective transverse-relaxation-optimized spectroscopy (IS-TROSY) is reported. The method considers aspects of both the NMR sample preparation and the experimental design. First, the protein is dissolved in a buffer with 50%H2O/50%D2O in order to promote the population of semideuterated NHD isotopomers in side chain amides of Asn/Gln residues. Second, a 13C'-coupled 2D 15N-1H IS-TROSY spectrum provides a stereospecific distinction between the geminal protons in the E and Z configurations of the carboxyamide group. Third, a suite of IS-TROSY-based triple-resonance NMR experiments, e.g. 3D IS-TROSY-HNCA and 3D IS-TROSY-HNCACB, are designed to correlate aliphatic carbon atoms with backbone amides and, for Asn/Gln residues, at the same time with side chain amides. The NMR assignment procedure is similar to that for small proteins using conventional 3D HNCA/3D HNCACB spectra, in which, however, signals from NH2 groups are often very weak or even missing due to the use of broad-band proton decoupling schemes and NOE data have to be used as a remedy. For large proteins, the use of conventional TROSY experiments makes resonances of side chain amides not observable at all. The application of IS-TROSY experiments to the 35-kDa yeast cytosine deaminase has established a complete resonance assignment for the backbone and stereospecific assignment for side chain amides, which otherwise could not be achieved with existing NMR experiments. Thus, the development of IS-TROSY-based method provides new opportunities for the NMR study of important structural and biological roles of carboxyamides and side chain moieties of arginine and lysine residues in large proteins as well as amino moieties in nucleic acids.
    Journal of Biomolecular NMR 01/2007; 36(4):205-14. · 2.85 Impact Factor
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    Lishan Yao, Honggao Yan, Robert I Cukier
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    ABSTRACT: A QM/MM method that combines ONIOM quantum chemistry and molecular dynamics is developed and applied to a step in the deamination of cytosine to uracil in yeast cytosine deaminase (yCD). A two-layer ONIOM calculation is used for the reaction complex, with an inner part treated at a high level for the chemical reaction (bond breaking) and a middle part treated at a lower level for relevant protein residues that are frozen in the quantum optimization. An outer layer (protein and solvent) is treated using MD. Configurations for the entire system are generated using MD and optimized with ONIOM. The method permits the use of high-level quantum calculations along with sufficient configurational sampling to approximate the potential of mean force for certain bond-breaking reactions. A previously proposed reaction mechanism for deamination (Sklenak, S.; Yao, L. S.; Cukier, R. I.; Yan, H. G. J. Am. Chem. Soc. 2004, 126, 14879) requires breaking the bond between a catalytic zinc and the O4 of uracil in order to permit product release. Using an ONIOM approach, direct bond cleavage was found to be energetically unfavorable. In the work presented here, the combined ONIOM MD method is used to show that the barrier for bond cleavage is small, approximately 3 kcal/mol, and, consequently, should not be the rate-limiting step in the reaction.
    The Journal of Physical Chemistry B 01/2007; 110(51):26320-6. · 3.61 Impact Factor

Publication Stats

288 Citations
173.04 Total Impact Points

Institutions

  • 1999–2014
    • Michigan State University
      • • Department of Biochemistry and Molecular Biology
      • • Department of Chemistry
      East Lansing, Michigan, United States
  • 2002–2012
    • National Cancer Institute (USA)
      • Macromolecular Crystallography Laboratory
      Maryland, United States
  • 2007
    • International Union of Toxicology
      Reston, Virginia, United States