A glutamine 67--> histidine mutation in homotetrameric R67 dihydrofolate reductase results in four mutations per single active site pore and causes substantial substrate and cofactor inhibition

R67 dihydrofolate reductase (DHFR) is a type II DHFR produced by bacteria as a resistance mechanism to increasing clinical use of the antibacterial drug trimethoprim. Type II DHFRs are not homologous in either sequence or structure with chromosomal DHFRs. The crystal structure of R67 DHFR shows a single active site pore that spans the length of the homotetramer. Related sites (due to a 222 symmetry element at the center of the pore) are used to bind ligands, i.e. each half of the pore can accommodate either the substrate, dihydrofolate (DHF), or the cofactor, NADPH, although DHF and NADPH are bound differently. To evaluate the role of glutamine 67 (and its symmetry- related Q167, Q267 and Q367 residues which occur at the center of the active site pore), a Q67H mutation was constructed. Binary binding of dihydrofolate (DHF; monitored by isothermal titration calorimetry) displays two identical sites with a Kd value of 0.04 microM, while binding of NADPH shows two sites possessing negative cooperativity with Kd values of 0.027 and 0.62 microM. A comparison of ligand binding in Q67H versus wild-type (wt) R67 DHFR indicates both ligands bind more tightly (80-6000-fold) and DHF binding in Q67H R67 DHFR no longer displays positive cooperativity as seen in wt R67 DHFR. Ternary complex binding in the Q67H mutant indicates a total of two ligands can bind per pore. Substantial substrate and cofactor inhibition are observed during catalysis, consistent with non-productive binding of either two DHF or two NADPH molecules in Q67H R67 DHFR. Because of the symmetry- related binding sites in the active site pore, the accumulation of potentially positive mutations in R67 DHFR is limited by the balance between tighter binding of ligands (and thus potentially increased catalytic efficiency) and inhibition that arises upon tighter binding of two identical ligands at symmetry-related sites.      

Improving protein solubility through rationally designed amino acid replacements: solubilization of the trimethoprim-resistant type S1 dihydrofolate reductase

In recent years resistance to the antibacterial agent trimethoprim(Tmp) has become more widespread and several Tmp-resistant (Tmp^rdihydrofolate reductases (DHFRs) have been described from Gram-negativebacteria. In staphylococci, however, only one Tmp^r DHFR (typeS1 DHFR) has been found so far, and this is located on transposonTn4003. To help understand the mechanism of resistance, we areinterested in determining the 3-D structure of the recombinantenzyme produced in Escherichia coli. However, the productionlevel of the type S1 DHFR was very low and >95% of the totalrecombinant protein accumulated in inclusion bodies. Furthermore,as a result of an internal start of translation, a truncatedderivative of the enzyme that copurified with the full-lengthenzyme was produced. We were able to increase the expressionlevel 20-fold by changing 18 N-terminal codons and to eliminatethe internal start of translation. In addition, through molecularmodelling and subsequent site-directed mutagenesis to replacetwo amino acids, we constructed a biochemically similar butsoluble derivative of the type SI DHFR that, after productionin E.coli, resulted in a 264-fold increase in DHFR activity.The highly overproduced enzyme was purified to homogeneity,characterized biochemically and crystallized.     

Slipknot or Crystallographic Error: A Computational Analysis of the Plasmodium falciparum DHFR Structural Folds

The presence of protein structures with atypical folds in the Protein Data Bank (PDB) is rare and may result from naturally occurring knots or crystallographic errors. Proper characterisation of such folds is imperative to understanding the basis of naturally existing knots and correcting crystallographic errors. If left uncorrected, such errors can frustrate downstream experiments that depend on the structures containing them. An atypical fold has been identified in P. falciparum dihydrofolate reductase (PfDHFR) between residues 20–51 (loop 1) and residues 191–205 (loop 2). This enzyme is key to drug discovery efforts in the parasite, necessitating a thorough characterisation of these folds. Using multiple sequence alignments (MSA), a unique insert was identified in loop 1 that exacerbates the appearance of the atypical fold-giving it a slipknot-like topology. However, PfDHFR has not been deposited in the knotted proteins database, and processing its structure failed to identify any knots within its folds. The application of protein homology modelling and molecular dynamics simulations on the DHFR domain of P. falciparum and those of two other organisms (E. coli and M. tuberculosis) that were used as molecular replacement templates in solving the PfDHFR structure revealed plausible unentangled or open conformations of these loops. These results will serve as guides for crystallographic experiments to provide further insights into the atypical folds identified.     

Cryo-kinetics Reveal Dynamic Effects on the Chemistry of Human Dihydrofolate Reductase

Effects of isotopic substitution on the rate constants of human dihydrofolate reductase (HsDHFR), an important target for anti-cancer drugs, have not previously been characterized due to its complex fast kinetics. Here, we report the results of cryo-measurements of the kinetics of the HsDHFR catalyzed reaction and the effects of protein motion on catalysis. Isotopic enzyme labeling revealed an enzyme KIE (k_H^LE/k_H^HE) close to unity above 0 °C; however, the enzyme KIE was increased to 1.72±0.15 at −20 °C, indicating that the coupling of protein motions to the chemical step is minimized under optimal conditions but enhanced at non-physiological temperatures. The presented cryogenic approach provides an opportunity to probe the kinetics of mammalian DHFRs, thereby laying the foundation for characterizing their transition state structure.     

The structural basis for the altered substrate specificity of the R292D active site mutant of aspartate aminotransferase from E.coli

Two refined crystal structures of aspartate aminotransferasefrom E.coli are reported. The wild type enzyme is in the pyridoxalphosphate (PLP) form and its structure has been determined to2.4 Å resolution, refined to an R-factor of 23.2%. Thestructure of the Arg292Asp mutant has been determined at 2.8Å resolution, refined to an R–factor of 20.3%. Thewild type and mutant crystals are isomorphous and the two structuresare very similar, with only minor changes in positions of importantactive site residues. As residue Arg292 is primarily responsiblefor the substrate charge specificity in the wild type enzyme,the mutant containing a charge reversal at this position mightbe expected to catalyze transamination of arginine as efficientlyas the wild type enzyme effects transamination of aspartate[Cronin,C.N. and Kirsch,J.F. (1988) Biochemistry, 27, 4572–4579].This mutant does in fact prefer arginine over aspartate as asubstrate, however, the rate of catalysis is much slower thanthat of the wild type enzyme with its physiological substrate,aspartate. A comparison of these two structures indicates thatthe poorer catalytic efficiency of R292D, when presented witharginine, is not due to a gross conformational difference, butis rather a consequence of both small side chain and main chainreorientations and the pre–existing active site polarenvironment, which greatly favors the wild type ion pair interaction.     

Phosphorothiolate analogues of phosphatidylinositols as assay substrates for phospholipase C

Accurate measurement of phosphatidylinositol-specific phospholipase C (PI-PLC) activity is important in view of the key role of this enzyme in signal-transduction pathways. In this work we synthesized enantiomerically pure phosphorothiolate analogues of all natural PI-PLC substrates, including those of phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2), 4-phosphate (PI-4-P), 5-phosphate (PI-5-P) and unphosphorylated PI, in both long- and short-chain versions. The enzymatic cleavage of these substrates produces thiol analogues of diacyl glycerol, which can be quantified by UV absorbance after treatment with dipyridyl disulfide. The monodisperse dihexanoyl derivatives are suitable substrates for PI-PLC assay: they give rise to high enzyme activity, and provide excellent linear kinetic responses. For all substrates, we found a good linear correlation between the reaction rate and the amount of enzyme; this indicated the suitability of this assay for enzyme quantification. The short-chain substrates enable the enzyme specificity with variously phosphorylated inositol head groups to be established--unobstructed by substrate aggregation, "scooting" kinetics on micelles, or surface dilution effects. The kinetic results indicated allosteric behavior of PLC for all substrates tested. We found that substrates phosphorylated at the inositol 4-position (phosphorothiolate analogues of PI-4,5-P2 and PI-4-P) displayed very similar kinetic properties, and were cleaved with approximately 20- to 30-fold higher activity than the 4-nonphosphorylated substrates (analogues of PI-5-P and PI). Hence it appears that interactions between the enzyme and the 4-phosphate group of the substrate, but not its 5-phosphate group, is important for PI-PLC catalysis. In addition, the binding affinities of all four substrate types were found to be quite similar; this indicates that the energy of enzyme interaction with the 4-phosphate group is directed almost entirely to catalysis.     

Binding of Natural and Synthetic Polyphenols to Human Dihydrofolate Reductase

Dihydrofolate reductase (DHFR) is the subject of intensive investigation since it appears to be the primary target enzyme for antifolate drugs. Fluorescence quenching experiments show that the ester bond-containing tea polyphenols (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) are potent inhibitors of DHFR with dissociation constants (K_D)of 0.9 and 1.8 μM, respectively, while polyphenols lacking the ester bound gallate moiety [e.g., (-)-epigallocatechin (EGC) and (-)-epicatechin (EC)] did not bind to this enzyme. To avoid stability and bioavailability problems associated with tea catechins we synthesized a methylated derivative of ECG (3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin; TMECG), which effectively binds to DHFR (K_D = 2.1 μM). In alkaline solution, TMECG generates a stable quinone methide product that strongly binds to the enzyme with a K_D of 8.2 nM. Quercetin glucuronides also bind to DHFR but its effective binding was highly dependent of the sugar residue, with quercetin-3-xyloside being the stronger inhibitor of the enzyme with a K_D of 0.6 μM. The finding that natural polyphenols are good inhibitors of human DHFR could explain the epidemiological data on their prophylactic effects for certain forms of cancer and open a possibility for the use of natural and synthetic polyphenols in cancer chemotherapy.     

Artificial Light Regulation of an Allosteric Bienzyme Complex by a Photosensitive Ligand

The artificial regulation of proteins by light is an emerging subdiscipline of synthetic biology. Here, we used this concept to photocontrol both catalysis and allostery within the heterodimeric enzyme complex imidazole glycerol phosphate synthase (ImGP‐S). ImGP‐S consists of the cyclase subunit HisF and the glutaminase subunit HisH, which is allosterically stimulated by substrate binding to HisF. We show that a light‐sensitive diarylethene (1,2‐dithienylethene, DTE)‐based competitive inhibitor in its ring‐open state binds with low micromolar affinity to the cyclase subunit and displaces its substrate from the active site. As a consequence, catalysis by HisF and allosteric stimulation of HisH are impaired. Following UV‐light irradiation, the DTE ligand adopts its ring‐closed state and loses affinity for HisF, restoring activity and allostery. Our approach allows for the switching of ImGP‐S activity and allostery during catalysis and appears to be generally applicable for the light regulation of other multienzyme complexes.     

Isopenicillin N Synthase: Crystallographic Studies

Isopenicillin N synthase (IPNS) is a non-heme iron oxidase (NHIO) that catalyses the cyclisation of tripeptide δ-(l -α-aminoadipoyl)-l -cysteinyl-d -valine (ACV) to bicyclic isopenicillin N (IPN). Over the last 25 years, crystallography has shed considerable light on the mechanism of IPNS catalysis. The first crystal structure, for apo-IPNS with Mn bound in place of Fe at the active site, reported in 1995, was also the first structure for a member of the wider NHIO family. This was followed by the anaerobic enzyme-substrate complex IPNS−Fe−ACV (1997), this complex plus nitric oxide as a surrogate for co-substrate dioxygen (1997), and an enzyme product complex (1999). Since then, crystallography has been used to probe many aspects of the IPNS reaction mechanism, by crystallising the protein with a diversity of substrate analogues and triggering the oxidative reaction by using elevated oxygen pressures to force the gaseous co-substrate throughout protein crystals and maximise synchronicity of turnover in crystallo. In this way, X-ray structures have been elucidated for a range of complexes closely related to and/or directly derived from key intermediates in the catalytic cycle, thereby answering numerous mechanistic questions that had arisen from solution-phase experiments, and posing many new ones. The results of these crystallographic studies have, in turn, informed computational experiments that have brought further insight. These combined crystallographic and computational investigations augment and extend the results of earlier spectroscopic analyses and solution phase studies of IPNS turnover, to enrich our understanding of this important protein and the wider NHIO enzyme family.     

Immobilization of lipase onto metal–organic frameworks for enantioselective hydrolysis and transesterification

Enzyme immobilization enhances the catalytic activity and stability of the enzyme, and also improves reusability. Metal–organic frameworks (MOFs), which possess diversified structures and porosity, have been used as excellent carriers for enzyme immobilization. Pseudomonas fluorescens lipase (PFL) has been successfully immobilized onto MOFs by covalent cross-linking to obtain a series of immobilized lipase (PFL@MOFs). PFL@MOFs are used for catalytic enantioselective hydrolysis of 2-(4-hydroxyphenyl) propionic acid ethyl ester enantiomers (2-HPPAEE) in aqueous medium and transesterification of 4-methoxymandelic acid enantiomers (4-MMA) in organic medium. The experimental results indicated that PFL@Uio-66(Zr) exhibits excellent enzymatic catalysis performances and high enantioselectives. In addition, to improve catalytic activity and reusability, PFL is modified by the polyethylene glycol (PEG) to prepare PEG-modified lipase (PFL-PEG), then PFL-PEG is immobilized onto Uio-66(Zr) to prepare PFL-PEG@Uio-66(Zr), demonstrating better reusability and catalytic activity compared with PFL@Uio-66(Zr).     

Reversible inactivation of the CG specific SssI DNA (cytosine-C5)-methyltransferase with a photocleavable protecting group

Caging of proteins by conjugation with a photocleavable group is a powerful approach for reversibly blocking enzymatic activity. Here we describe the covalent modification of the bacterial SssI DNA methyltransferase (M.SssI) with the cysteine-specific reagent 4,5-dimethoxy-2-nitrobenzylbromide (DMNBB). M.SssI contains two cysteine residues; replacement of the active-site Cys141 with Ser resulted in an approximately 100-fold loss of enzymatic activity; this indicates an important role for this residue in catalysis. However, replacement of Cys368 with Ala did not affect methyltransferase activity. Treatment of the Cys368Ala mutant enzyme with DMNBB led to an almost complete loss of activity. Irradiation of the inactivated enzyme with near-ultraviolet light (320-400 nm) restored 60 % of the catalytic activity. This indicates that caging by DMNBB can be used for the reversible inactivation of M.SssI.     

Are the Catalytic Properties of Enzymes from Piezophilic Organisms Pressure Adapted?

We report the crystal structure of dihydrofolate reductase (DHFR) from the psychropiezophilic bacterium Moritella profunda, which was isolated from the deep ocean at 2 °C and 280 bar. The structure is typical of a chromosomal DHFR and we were unable to identify any obvious structural features that would suggest pressure adaptation. In particular, the core regions of the enzyme are virtually identical to those of the DHFR from the mesophile Escherichia coli. The steady‐state rate at pH 9, which is limited by hydride transfer at atmospheric pressure, is roughly constant between 1 and 750 bar, falling at higher pressures. However, the value of K_M increases with increasing pressure, and as a result k_cat/K_M decreases over the entire pressure range studied. Isotope effect studies showed that increasing the pressure causes a change in the rate‐limiting step of the reaction. We therefore see no evidence of pressure adaptation in either the structure or the activity of this enzyme.     

纳米酶催化剂制备方法研究进展

酶催化具有化学、区位和立体选择性,这使得其成为绿色合成化学品的理想催化剂。然而,天然酶常因其在工业催化条件下的活性和稳定性较低而难以使用。纳米技术为构建高效酶催化剂提供了新的可能性。通过简便、高效、低成本的方法制备出具有高催化活性和高稳定性的纳米酶催化剂,同时提高纳米酶催化剂的可操作性和可回收重复使用特性是其中的关键问题。介绍了纳米酶催化剂的研究现状和制备方法,重点介绍了采用共沉淀方法制备酶-无机晶体杂化纳米催化剂、酶-金属有机骨架材料杂化纳米催化剂,以及制备具有温度和磁响应特性的纳米酶催化剂,并对纳米酶催化剂在酶催化合成医药化学品方面的应用前景进行了探讨。     

Site-directed alteration of Glu197 and Glu66 in a pyruvoyl-dependent histidine decarboxylase

Site-directed mutagenesis has been used to explore the roleof two carboxylates in the active site of histidine decarboxylasefrom Lactobacillus 30a. The most striking observation is thatconversion of Glu197 to either Gln or Asp causes a major decreasein catalytic rate while enhancing substrate binding. This isconsistent with models based on X-ray diffraction results whichsuggest that the acid may protonate a reaction intermediateduring catalysis. The Asp197 protein undergoes a suicide reactionwith substrate, apparently triggered by inappropriate protonationof the intermediate. This leads to decarboxylation-dependenttransamination which converts the pyruvoyl cofactor to an alanine,inactivating the enzyme. Conversion of Glu66 to Gln affectsparameters of kinetic cooperativity. The mutation fixes theHill number at – 1.5, midway between the pH-dependentvalues of the wild-type enzyme.     

A Comparison of the Binding of the Ligand Trimethoprim to Bacterial and Vertebrate Dihydrofolate Reductases

In this paper, we report on studies of ligand binding to the enzyme dihydrofolate reductase (DHFR). Energy minimizations of four complexes of DHFR with the inhibitor trimethoprim, an antibiotic, and the cofactor NADPH have been carried out in order to investigate the energetics responsible for the 100,000-fold increase in binding affinity of trimethoprim to E. coli DHFR compared with chicken liver DHFR. Several factors suggested to be responsible for the enhanced binding in bacterial DHFR's were investigated in terms of intermolecular and intramolecular energetics. The strain energies of trimethoprim in the four complexes were calculated and found to be about 6 kcal mol⁻¹ in all complexes of the two species. In the binary complex of chicken liver DHFR, where the largest variation was observed, 2 kcal mol⁻¹ higher than in the other complexes, it was found that this increase was compensated for by the slightly more favorable intermolecular interaction of the trimethoxyphenyl moiety with the protein. Comparison of the minimized binary and ternary complexes of E. coli allowed us to investigate the cooperativity in the binding of trimethoprim and NADPH in the bacterial enzyme in terms of the underlying intermolecular forces. This cooperativity was found to be due to a direct trimethoprim - NADPH interaction in the E. coli enzyme rather than enhanced protein-inhibitor interactions induced upon binding of the cofactor. These interactions are not as favorable in the vertebrate enzyme, consistent with the significantly diminished cooperativity observed in this enzyme.     

Design of biomimetic catalysts by molecular imprinting in synthetic polymers: the role of transition state stabilization

The impressive efficiency and selectivity of biological catalysts has engendered a long-standing effort to understand the details of enzyme action. It is widely accepted that enzymes accelerate reactions through their steric and electronic complementarity to the reactants in the rate-determining transition states. Thus, tight binding to the transition state of a reactant (rather than to the corresponding substrate) lowers the activation energy of the reaction, providing strong catalytic activity. Debates concerning the fundamentals of enzyme catalysis continue, however, and non-natural enzyme mimics offer important additional insight in this area. Molecular structures that mimic enzymes through the design of a predetermined binding site that stabilizes the transition state of a desired reaction are invaluable in this regard. Catalytic antibodies, which can be quite active when raised against stable transition state analogues of the corresponding reaction, represent particularly successful examples. Recently, synthetic chemistry has begun to match nature's ability to produce antibody-like binding sites with high affinities for the transition state. Thus, synthetic, molecularly imprinted polymers have been engineered to provide enzyme-like specificity and activity, and they now represent a powerful tool for creating highly efficient catalysts. In this Account, we review recent efforts to develop enzyme models through the concept of transition state stabilization. In particular, models for carboxypeptidase A were prepared through the molecular imprinting of synthetic polymers. On the basis of successful experiments with phosphonic esters as templates to arrange amidinium groups in the active site, the method was further improved by combining the concept of transition state stabilization with the introduction of special catalytic moieties, such as metal ions in a defined orientation in the active site. In this way, the imprinted polymers were able to provide both an electrostatic stabilization for the transition state through the amidinium group as well as a synergism of transition state recognition and metal ion catalysis. The result was an excellent catalyst for carbonate hydrolysis. These enzyme mimics represent the most active catalysts ever prepared through the molecular imprinting strategy. Their catalytic activity, catalytic efficiency, and catalytic proficiency clearly surpass those of the corresponding catalytic antibodies. The active structures in natural enzymes evolve within soluble proteins, typically by the refining of the folding of one polypeptide chain. To incorporate these characteristics into synthetic polymers, we used the concept of transition state stabilization to develop soluble, nanosized carboxypeptidase A models using a new polymerization method we term the "post-dilution polymerization method". With this methodology, we were able to prepare soluble, highly cross-linked, single-molecule nanoparticles. These particles have controlled molecular weights (39 kDa, for example) and, on average, one catalytically active site per particle. Our strategies have made it possible to obtain efficient new enzyme models and further advance the structural and functional analogy with natural enzymes. Moreover, this bioinspired design based on molecular imprinting in synthetic polymers offers further support for the concept of transition state stabilization in catalysis.     

Helices on Interdomain Interface Couple Catalysis in the ATPPase Domain with Allostery in Plasmodium falciparum GMP Synthetase

GMP synthetase catalyses the conversion of XMP to GMP through a series of reactions that include hydrolysis of Gln to generate ammonia in the glutamine amidotransferase (GATase) domain, activation of XMP to adenyl-XMP intermediate in the ATP pyrophosphatase (ATPPase) domain and reaction of ammonia with the intermediate to generate GMP. The functioning of GMP synthetases entails bidirectional domain crosstalk, which leads to allosteric activation of the GATase domain, synchronization of catalytic events and tunnelling of ammonia. Herein, we have taken recourse to the analysis of structures of GMP synthetases, site-directed mutagenesis and steady-state and transient kinetics on the Plasmodium falciparum enzyme to decipher the molecular basis of catalysis in the ATPPase domain and domain crosstalk. Our results suggest an arrangement at the interdomain interface, of helices with residues that play roles in ATPPase catalysis as well as domain crosstalk enabling the coupling of ATPPase catalysis with GATase activation. Overall, the study enhances our understanding of GMP synthetases, which are drug targets in many infectious pathogens.     

Charged nucleobases and their potential for RNA catalysis

Catalysis in living cells is carried out by both proteins and RNA. Protein enzymes have been known for over 200 years, but RNA enzymes, or "ribozymes", were discovered only 30 years ago. Developing insight into RNA enzyme mechanisms is invaluable for better understanding both extant biological catalysis as well as the primitive catalysis envisioned in an early RNA-catalyzed life. Natural ribozymes include large RNAs such as the group I and II introns; small RNAs such as the hepatitis delta virus and the hairpin, hammerhead, VS, and glmS ribozymes; and the RNA portion of the ribosome and spliceosome. RNA enzymes use many of the same catalytic strategies as protein enzymes, but do so with much simpler side chains. Among these strategies are metal ion, general acid-base, and electrostatic catalysis. In this Account, we examine evidence for participation of charged nucleobases in RNA catalysis. Our overall approach is to integrate direct measurements on catalytic RNAs with thermodynamic studies on oligonucleotide model systems. The charged amino acids make critical contributions to the mechanisms of nearly all protein enzymes. Ionized nucleobases should be critical for RNA catalysis as well. Indeed, charged nucleobases have been implicated in RNA catalysis as general acid-bases and oxyanion holes. We provide an overview of ribozyme studies involving nucleobase catalysis and the complications involved in developing these mechanisms. We also consider driving forces for perturbation of the pK(a) values of the bases. Mechanisms for pK(a) values shifting toward neutrality involve electrostatic stabilization and the addition of hydrogen bonding. Both mechanisms couple protonation with RNA folding, which we treat with a thermodynamic formalism and conceptual models. Furthermore, ribozyme reaction mechanisms can be multichannel, which demonstrates the versatility of ribozymes but makes analysis of experimental data challenging. We examine advances in measuring and analyzing perturbed pK(a) values in RNA. Raman crystallography and fluorescence spectroscopy have been especially important for pK(a) measurement. These methods reveal pK(a) values for the nucleobases A or C equal to or greater than neutrality, conferring potential histidine- and lysine/arginine-like behavior on them. Structural support for ionization of the nucleobases also exists: an analysis of RNA structures in the databases conducted herein suggests that charging of the bases is neither especially uncommon nor difficult to achieve under cellular conditions. Our major conclusions are that cationic and anionic charge states of the nucleobases occur in RNA enzymes and that these states make important catalytic contributions to ribozyme activity. We conclude by considering outstanding questions and possible experimental and theoretical approaches for further advances.