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author:("Yan, hongqiao")
1.  Structure of Bacterial Transcription Factor SpoIIID and Evidence for a Novel Mode of DNA Binding 
Journal of Bacteriology  2014;196(12):2131-2142.
SpoIIID is evolutionarily conserved in endospore-forming bacteria, and it activates or represses many genes during sporulation of Bacillus subtilis. An 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 nuclear magnetic resonance (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 of 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.
PMCID: PMC4054184  PMID: 24584501
2.  Crystallographic and molecular dynamics simulation analysis of Escherichia coli dihydroneopterin aldolase 
Cell & Bioscience  2014;4(1):52.
Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin and also the epimerization of DHNP to 7,8-dihydromonapterin. Previously, we determined the crystal structure of Staphylococcus aureus DHNA (SaDHNA) in complex with the substrate analogue neopterin (NP). We also showed that Escherichia coli DHNA (EcDHNA) and SaDHNA have significantly different binding and catalytic properties by biochemical analysis. On the basis of these structural and functional data, we proposed a catalytic mechanism involving two proton wires.
To understand the structural basis for the biochemical differences and further investigate the catalytic mechanism of DHNA, we have determined the structure of EcDHNA complexed with NP at 1.07-Å resolution [PDB:2O90], built an atomic model of EcDHNA complexed with the substrate DHNP, and performed molecular dynamics (MD) simulation analysis of the substrate complex. EcDHNA has the same fold as SaDHNA and also forms an octamer that consists of two tetramers, but the packing of one tetramer with the other is significantly different between the two enzymes. Furthermore, the structures reveal significant differences in the vicinity of the active site, particularly in the loop that connects strands β3 and β4, mainly due to the substitution of nearby residues. The building of an atomic model of the complex of EcDHNA and the substrate DHNP and the MD simulation of the complex show that some of the hydrogen bonds between the substrate and the enzyme are persistent, whereas others are transient. The substrate binding model and MD simulation provide the molecular basis for the biochemical behaviors of the enzyme, including noncooperative substrate binding, indiscrimination of a pair of epimers as the substrates, proton wire switching during catalysis, and formation of epimerization product.
The EcDHNA and SaDHNA structures, each in complex with NP, reveal the basis for the biochemical differences between EcDHNA and SaDHNA. The atomic substrate binding model and MD simulation offer insights into substrate binding and catalysis by DHNA. The EcDHNA structure also affords an opportunity to develop antimicrobials specific for Gram-negative bacteria, as DHNAs from Gram-negative bacteria are highly homologous and E. coli is a representative of this class of bacteria.
PMCID: PMC4176595  PMID: 25264482
Dihydroneopterin aldolase; DHNA; Structure; Dynamics; Catalysis
3.  Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: New lead exhibits a distinct binding mode 
Bioorganic & medicinal chemistry  2012;20(14):4303-4309.
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 analogue 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.
PMCID: PMC3389233  PMID: 22727779
Antibacterial; Bisubstrate; Folate; HPPK; Pterin
4.  Role of Glutamate 64 in the Activation of the Prodrug 5-fluorocytosine by Yeast Cytosine Deaminase† 
Biochemistry  2011;51(1):475-486.
Yeast cytosine deaminase 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, NMR, and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in kcat and a dramatic increase in Km, indicating Glu64 is important for both binding and catalysis in the activation of 5FC. 19F-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. 1H and 15N 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 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 1H and 15N NMR analysis. To explore the functional role of Glu64 in catalysis, deamination of cytosine catalyzed by the E64A mutant was investigated 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 binding and chemical transformation in the conversion of the prodrug 5FC to the anticancer drug 5-fluorouracil.
PMCID: PMC3272785  PMID: 22208667
5.  Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: New design with improved properties 
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 Kd and IC50 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.
PMCID: PMC3257516  PMID: 22169600
Antibacterial; Bisubstrate; Folate; HPPK; Pterin
6.  Role of Protein Conformational Dynamics in the Catalysis by 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase† 
Protein and peptide letters  2011;18(4):328-335.
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.
PMCID: PMC3482336  PMID: 21222642
Bisubstrate enzyme; enzymatic catalysis; folate biosynthesis; 6-hydroxymethyl-7; 8-dihydropterin pyrophosphokinase; HPPK; NMR; protein dynamics; X-ray crystallography
7.  Probing Single-Molecule Enzyme Active-Site Conformational State Intermittent Coherence 
Journal of the American Chemical Society  2011;133(36):14389-14395.
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 multi-step 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.
PMCID: PMC3198842  PMID: 21823644
8.  TROSY of side-chain amides in large proteins 
Journal of Magnetic Resonance  2007;186(2):319-326.
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–HE{DZ} and 15N–HZ{DE} 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–HE{DZ} isotopomers of side-chain amides are as significant as on backbone amides.
PMCID: PMC3272764  PMID: 17347000
TROSY; NMR spectroscopy; Sensitivity enhancement; Isotopomer selectivity; Stereospecific NMR assignment; Side-chain amides
9.  Mechanism of Dihydroneopterin Aldolase: The Functional Roles of the Conserved Active Site Glutamate and Lysine Residues† 
Biochemistry  2006;45(51):15232-15239.
Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) in the folate biosynthetic pathway. There are four conserved active site residues at the active site, E22, Y54, E74, and K100 in Staphylococcus aureus DHNA (SaDHNA), corresponding to E21, Y53, E73, and K98 in Escherichia coli DHNA (EcDHNA). The functional roles of the conserved glutamate and lysine residues have been investigated by site-directed mutagenesis in this work. E22 and E74 of SaDHNA and E21, E73, and K98 of EcDHNA were replaced by alanine. K100 of SaDHNA was replaced by alanine and glutamine. The mutant proteins were characterized by equilibrium binding, stopped-flow binding, and steady-state kinetic analyses. For SaDHNA, none of the mutations except E74A caused dramatic changes in the affinities of the enzyme for the substrate or product analogues or the rate constants. The Kd values for SaE74A were estimated to be >3000 μM, suggesting that the Kd values of the mutant is at least 100 times those of the wild-type enzyme. For EcDHNA, the E73A mutation caused increases in the Kd values for the substrate or product analogues neopterin (MP), monapterin (NP), and 6-hydroxypterin (HPO) by factors of 340, 160, and 5600, respectively, relative to those of the wild-type enzyme. The K98A mutation caused increases in the Kd values for NP, MP, and HPO by factors of 14, 3.6, and 230, respectively. The E21A mutation caused increases in the Kd values for NP and HPO by factors of 2.2 and 42, respectively, but a decrease in the Kd value for MP by a factor of 3.3. The E22 (E21) and K100 (K98) mutations caused decreases in the kcat values by factors of 1.3×104 to 2×104. The E74 (E73) mutation caused decreases in the kcat values by factors of ~10. The results suggested that E74 of SaDHNA and E73 of EcDHNA are important for substrate binding, but their roles in catalysis are minor. In contrast, E22 and K100 of SaDHNA are important for catalysis, but their roles in substrate binding are minor. On the other hand, E21 and K98 of EcDHNA are important for both substrate binding and catalysis.
PMCID: PMC3018710  PMID: 17176045
10.  Hydrogen-Bond Detection, Configuration Assignment, and Rotamer Correction of Side-Chain Amides in Large Proteins by NMR through Protium/Deuterium Isotope Effects 
In this study, the configuration and hydrogen-bonding network of side-chain amides in a 35 kDa protein are determined via measuring differential and across-hydrogen-bond H/D isotope effects by the IS-TROSY technique, which leads to a reliable recognition and correction of erroneous rotamers frequently found in protein structures. First, the differential two-bond isotope effects on carbonyl 13C′ shifts, defined as Δ2Δ13C′ (ND) = 2Δ13C′ (NDE) − 2Δ13C′ (NDZ), provide a reliable means for the configuration assignment for side-chain amides, as environmental effects (hydrogen bonds and charges etc.) are greatly attenuated over the two bonds separating 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 15N chemical shifts, defined as Δ1Δ15N(D) = 1Δ15N(DE) − 1Δ15N(DZ), 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 across-H-bond H/D isotope effects can be mapped onto more complicated hydrogen-bonding networks involving bifurcated hydrogen-bonds. Third, the correlations between Δ1Δ15N(D) and hydrogen-bonding interactions afford an effective means for the correction of erroneous rotamer assignments of side-chain amides. The rotamer correction via differential isotope effects is not only robust but simple and can be applied to large proteins.
PMCID: PMC3018730  PMID: 18973166
Configuration determination; hydrogen bonding; isotope effects; NMR spectroscopy; protein side-chain amide; rotamer assignment
11.  Productive versus Unproductive Nucleotide Binding in Yeast Guanylate Kinase Mutants: Comparison of R41M with K14M by Proton Two Dimensional Transferred NOESY† 
Biochemistry  2009;48(24):5532-5540.
The R41M and K14M mutant enzymes of yeast guanylate kinase (GKy) were studied to investigate the effects of these site-directed mutations on bound-substrate conformations. Published X-ray crystal structures of yeast guanylate kinase indicate that K14 is part of the “ P ” loop involved in ATP and ADP binding while R41 is suggested as a hydrogen bonding partner for the phosphoryl moiety of GMP. Both of these residues might be involved in transition state stabilization. Adenosine conformations of ATP and ADP and guanosine conformations of GMP bound to R41M and K14M mutant yeast guanylate kinase in the complexes GKy•MgATP, GKy•MgADP, and GKy•MgADP•[u-13C]GMP were determined by two-dimensional transferred nuclear Overhauser effect (TRNOESY) measurements combined with molecular dynamics simulations, and these conformations were compared with previously published conformations for the wild type. In the fully constrained, two substrate complexes, GKy•MgADP•[u-13C]GMP, the guanyl glycosidic torsion angle, χ, is 51±5° for R41M and 47±5° for K14M. Both of which are similar to the published 50±5° published for wild type. For R41M with adenyl nucleotides, the glycosidic torsion angle, χ, was 55±5° with MgATP, and 47±5° with MgADP, which compares well to the 54±5° published for wild type. However, for K14M with adenyl nucleotides, the glycosidic torsion angle was 30±5° with MgATP and 28±5° with MgADP. The results indicate that bound adenyl-nucleotides have significantly different conformations in the wild-type and K14M mutant enzymes, suggesting that K14 plays an important role in orienting the triphosphate of MgATP for catalysis.
PMCID: PMC2772131  PMID: 19419194
12.  Structural Basis for the Aldolase and Epimerase Activities of Staphylococcus aureus Dihydroneopterin Aldolase 
Journal of molecular biology  2007;368(1):161-169.
Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and also 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.
PMCID: PMC1885205  PMID: 17331536
aldolase; dihydroneopterin aldolase; dihydroneopterin; dihydromonapterin; pterin

Results 1-12 (12)