Characterization and crystal structure of lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase (cDHDPS) protein

The lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase enzyme (cDHDPS) was recently successfully introduced into maize plants to enhance the level of lysine in the grain. To better understand lysine insensitivity of the cDHDPS, we expressed, purified, kinetically characterized the protein, and solved its X-ray crystal structure. The cDHDPS enzyme has a fold and overall structure that is highly similar to other DHDPS proteins. A noteworthy feature of the active site is the evidence that the catalytic lysine residue forms a Schiff base adduct with pyruvate. Analyses of the cDHDPS structure in the vicinity of the putative binding site for S-lysine revealed that the allosteric binding site in the Escherichia coli DHDPS protein does not exist in cDHDPS due to three non-conservative amino acids substitutions, and this is likely why cDHDPS is not feedback inhibited by lysine.     

Asparagine-84, a regulatory allosteric site residue,helps maintain the quaternary structure of Campylobacter jejuni dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS) from Campylobacter jejuni is a natively homotetrameric enzyme that catalyzes the first unique reaction of (S)-lysine biosynthesis and is feedback-regulated by lysine through binding to an allosteric site. High-resolution structures of the DHDPS-lysine complex have revealed significant insights into the binding events. One key asparagine residue, N84, makes hydrogen bonds with both the carboxyl and the α-amino group of the bound lysine. We generated two mutants, N84A and N84D, to study the effects of these changes on the allosteric site properties. However, under normal assay conditions, N84A displayed notably lower catalytic activity, and N84D showed no activity. Here we show that these mutations disrupt the quaternary structure of DHDPS in a concentration-dependent fashion, as demonstrated by size-exclusion chromatography, multi-angle light scattering, dynamic light scattering, small-angle X-ray scattering (SAXS) and high-resolution protein crystallography.     

Conserved main-chain peptide distortions: a proposed role for Ile203 in catalysis by dihydrodipicolinate synthase

In recent years, dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) has received considerable attention from a mechanistic and structural viewpoint. DHDPS catalyzes the reaction of (S)-aspartate-beta-semialdehyde with pyruvate, which is bound via a Schiff base to a conserved active-site lysine (Lys161 in the enzyme from Escherichia coli). To probe the mechanism of DHDPS, we have studied the inhibition of E. coli DHDPS by the substrate analog, beta-hydroxypyruvate. The K (i) was determined to be 0.21 (+/-0.02) mM, similar to that of the allosteric inhibitor, (S)-lysine, and beta-hydroxypyruvate was observed to cause time-dependent inhibition. The inhibitory reaction with beta-hydroxypyruvate could be qualitatively followed by mass spectrometry, which showed initial noncovalent adduct formation, followed by the slow formation of the covalent adduct. It is unclear whether beta-hydroxypyruvate plays a role in regulating the biosynthesis of meso-diaminopimelate and (S)-lysine in E. coli, although we note that it is present in vivo. The crystal structure of DHDPS complexed with beta-hydroxypyruvate was solved. The active site clearly showed the presence of the inhibitor covalently bound to the Lys161. Interestingly, the hydroxyl group of beta-hydroxypyruvate was hydrogen-bonded to the main-chain carbonyl of Ile203. This provides insight into the possible catalytic role played by this peptide unit, which has a highly strained torsion angle (omega approximately 201 degrees ). A survey of the known DHDPS structures from other organisms shows this distortion to be a highly conserved feature of the DHDPS active site, and we propose that this peptide unit plays a critical role in catalysis.     

Functional rescue of a bacterial dapA auxotroph with a plant cDNA library selects for mutant clones encoding a feedback-insensitive dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS; EC4.2.1.52) catalyses the first reaction of lysine biosynthesis in plants and bacteria. Plant DHDPS enzymes are strongly inhibited by lysine (I0.5 approximately 10 microM), whereas the bacterial enzymes are less (50-fold) or insensitive to lysine inhibition. We found that plant dhdps sequences expressing lysine-sensitive DHDPS enzymes are unable to complement a bacterial auxotroph, although a functional plant DHDPS enzyme is formed. As a consequence of this, plant dhdps cDNA clones which have been isolated through functional complementation using the DHDPS-deficient Escherichia coli strain encode mutated DHDPS enzymes impaired in lysine inhibition. The experiments outlined in this article emphasize that heterologous complementation can select for mutant clones when altered protein properties are requisite for functional rescue. In addition, the mutants rescued by heterologous complementation revealed a new critical amino acid substitution which renders lysine insensitivity to the plant DHDPS enzyme. An interpretation is given for the impaired inhibition mechanism of the mutant DHDPS enzyme by integrating the identified amino acid substitution in the DHDPS protein structure.     

Dihydrodipicolinate synthase (DHDPS) from Escherichia coli displays partial mixed inhibition with respect to its first substrate, pyruvate

Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) mediates the first unique reaction of (S)-lysine biosynthesis in plants and microbes-the condensation of (S)-aspartate-beta-semialdehyde ((S)-ASA) and pyruvate. It has been shown that DHDPS is partially feedback inhibited by (S)-lysine; it is suggested that this mechanism regulates flux through the DAP biosynthetic pathway. Others have characterised DHDPS from Escherichia coli with respect to (S)-lysine inhibition. They have concluded that, with respect to pyruvate, the first substrate of the reaction, DHDPS shows uncompetitive inhibition: as such, they further suggest that (S)-lysine inhibits DHDPS via interaction with the binding site for the second substrate, (S)-ASA. Yet, this finding is based on the assumption that (S)-lysine is a fully uncompetitive inhibitor. In light of crystallographic studies, which lead to the proposal that (S)-lysine affects the putative proton-relay of DHDPS, we re-evaluated the inhibition mechanism of DHDPS with respect to (S)-lysine by incorporating the observed hyperbolic inhibition. Our data showed that lysine is not an uncompetitive inhibitor, but a mixed inhibitor when pyruvate and (S)-lysine concentrations were varied. Thus, consistent with the crystallographic data, (S)-lysine must have an effect on the initial steps of the DHDPS reaction, including the binding of pyruvate and Schiff base formation.     

Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana

In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S)-lysine. Structural studies of At-DHDPS2 show (S)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.     

Engineering a feedback inhibition-insensitive plant dihydrodipicolinate synthase to increase lysine content in Camelina sativa seeds

Camelina sativa (camelina) is emerging as an alternative oilseed crop due to its short growing cycle, low input requirements, adaptability to less favorable growing environments and a seed oil profile suitable for biofuel and industrial applications. Camelina meal and oil are also registered for use in animal and fish feeds; however, like meals derived from most cereals and oilseeds, it is deficient in certain essential amino acids, such as lysine. In higher plants, the reaction catalyzed by dihydrodipicolinate synthase (DHDPS) is the first committed step in the biosynthesis of lysine and is subject to regulation by lysine through feedback inhibition. Here, we report enhancement of lysine content in C. sativa seed via expression of a feedback inhibition-insensitive form of DHDPS from Corynebacterium glutamicums (CgDHDPS). Two genes encoding C. sativa DHDPS were identified and the endogenous enzyme is partially insensitive to lysine inhibition. Site-directed mutagenesis was used to examine the impact of alterations, alone and in combination, present in lysine-desensitized DHDPS isoforms from Arabidopsis thaliana DHDPS (W53R), Nicotiana tabacum (N80I) and Zea mays (E84K) on C. sativa DHDPS lysine sensitivity. When introduced alone, each of the alterations decreased sensitivity to lysine; however, enzyme specific activity was also affected. There was evidence of molecular or structural interplay between residues within the C. sativa DHDPS allosteric site as coupling of the W53R mutation with the N80V mutation decreased lysine sensitivity of the latter, but not to the level with the W53R mutation alone. Furthermore, the activity and lysine sensitivity of the triple mutant (W53R/N80V/E84T) was similar to the W53R mutation alone or the C. glutamicum DHDPS. The most active and most lysine-insensitive C. sativa DHDPS variant (W53R) was not inhibited by free lysine up to 1 mM, comparable to the C. glutamicums enzyme. Seed lysine content increased 13.6 -22.6% in CgDHDPS transgenic lines and 7.6–13.2% in the mCsDHDPS lines. The high lysine-accumulating lines from this work may be used to produce superior quality animal feed with improved essential amino acid profile.

     

Crystal, solution and in silico structural studies of dihydrodipicolinate synthase from the common grapevine

Dihydrodipicolinate synthase (DHDPS) catalyzes the rate limiting step in lysine biosynthesis in bacteria and plants. The structure of DHDPS has been determined from several bacterial species and shown in most cases to form a homotetramer or dimer of dimers. However, only one plant DHDPS structure has been determined to date from the wild tobacco species, Nicotiana sylvestris (Blickling et al. (1997) J. Mol. Biol. 274, 608-621). Whilst N. sylvestris DHDPS also forms a homotetramer, the plant enzyme adopts a 'back-to-back' dimer of dimers compared to the 'head-to-head' architecture observed for bacterial DHDPS tetramers. This raises the question of whether the alternative quaternary architecture observed for N. sylvestris DHDPS is common to all plant DHDPS enzymes. Here, we describe the structure of DHDPS from the grapevine plant, Vitis vinifera, and show using analytical ultracentrifugation, small-angle X-ray scattering and X-ray crystallography that V. vinifera DHDPS forms a 'back-to-back' homotetramer, consistent with N. sylvestris DHDPS. This study is the first to demonstrate using both crystal and solution state measurements that DHDPS from the grapevine plant adopts an alternative tetrameric architecture to the bacterial form, which is important for optimizing protein dynamics as suggested by molecular dynamics simulations reported in this study.     

Characterization of a thermostable dihydrodipicolinate synthase from Thermoanaerobacter tengcongensis

Dihydrodipicolinate synthase (DHDPS) catalyses the first reaction of the (S)-lysine biosynthesis pathway in bacteria and plants. The hypothetical gene for dihydrodipicolinate synthase (dapA) of Thermoanaerobacter tengcongensis was found in a cluster containing several genes of the diaminopimelate lysine–synthesis pathway. The dapA gene was cloned in Escherichia coli, DHDPS was subsequently produced and purified to homogeneity. The T. tengcongensis DHDPS was found to be thermostable (T _0.5 = 3 h at 90°C). The specific condensation of pyruvate and (S)-aspartate-β -semialdehyde was catalyzed optimally at 80°C at pH 8.0. Enzyme kinetics were determined at 60°C, as close as possible to in vivo conditions. The established kinetic parameters were in the same range as for example E. coli dihydrodipicolinate synthase. The specific activity of the T. tengcongensis DHDPS was relatively high even at 30°C. Like most dihydrodipicolinate synthases known at present, the DHDPS of T. tengcongensis seems to be a tetramer. A structural model reveals that the active site is well conserved. The binding site of the allosteric inhibitor lysine appears not to be conserved, which agrees with the fact that the DHDPS of T. tengcongensis is not inhibited by lysine under physiological conditions.     

The crystal structure of three site-directed mutants of Escherichia coli dihydrodipicolinate synthase: further evidence for a catalytic triad

Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyses the branchpoint reaction of lysine biosynthesis in plants and microbes: the condensation of (S)-aspartate-beta-semialdehyde and pyruvate. The crystal structure of wild-type DHDPS has been published to 2.5A, revealing a tetrameric molecule comprised of four identical (beta/alpha)(8)-barrels, each containing one active site. Previous workers have hypothesised that the catalytic mechanism of the enzyme involves a catalytic triad of amino acid residues, Tyr133, Thr44 and Tyr107, which provide a proton shuttle to transport protons from the active site to solvent. We have tested this hypothesis using site-directed mutagenesis to produce three mutant enzymes: DHDPS-Y133F, DHDPS-T44V and DHDPS-Y107F. Each of these mutants has substantially reduced activity, consistent with the catalytic triad hypothesis. We have determined each mutant crystal structure to at least 2.35A resolution and compared the structures to the wild-type enzyme. All mutant enzymes crystallised in the same space group as the wild-type form and only minor differences in structure are observed. These results suggest that the catalytic triad is indeed in operation in wild-type DHDPS.     

The allosteric nature of substrate inhibition of rabbit skeletal muscle fructose-1,6-diphosphatase

The mechanism of substrate inhibition of rabbit skeletal muscle fructose-1.6-bisphosphatase was examined. Analysis of substrate saturation curves obtained at different concentrations of Mg2+ revealed that the inhibiting effect of the substrate is manifested only within the complex with Mg2+, whereas the free form of the substrate causes no inhibition. Evidence for the allosteric nature of substrate inhibition was obtained by partial desensitization of the enzyme in the presence of salicylates. It was shown that fructose-1.6-bisphosphatase inhibition by the substrate obeys positively cooperative kinetics and is noncompetitive with respect to the substrate involved in the catalytic process. Studies on enzyme modification in the presence of DTNB and pyridoxal-5'-phosphate demonstrated that the inhibiting concentrations of the substrate are bound to the center which differs from the allosteric site for AMP. It is suggested that the antagonism of simultaneous action of AMP and high substrate concentrations may be due to the competition of the phosphate groups of these ligands for binding to the common lysine residue located in the overlapping region of two allosteric sites.     

Identification of the bona fide DHDPS from a common plant pathogen

Agrobacterium tumefaciens is a Gram‐negative soil‐borne bacterium that causes Crown Gall disease in many economically important crops. The absence of a suitable chemical treatment means there is a need to discover new anti‐Crown Gall agents and also characterize bona fide drug targets. One such target is dihydrodipicolinate synthase (DHDPS), a homo‐tetrameric enzyme that catalyzes the committed step in the metabolic pathway yielding meso‐diaminopimelate and lysine. Interestingly, there are 10 putative DHDPS genes annotated in the A. tumefaciens genome, including three whose structures have recently been determined (PDB IDs: 3B4U, 2HMC, and 2R8W). However, we show using quantitative enzyme kinetic assays that nine of the 10 dapA gene products, including 3B4U, 2HMC, and 2R8W, lack DHDPS function in vitro. A sequence alignment showed that the product of the dapA7 gene contains all of the conserved residues known to be important for DHDPS catalysis and allostery. This gene was cloned and the recombinant product expressed and purified. Our studies show that the purified enzyme (i) possesses DHDPS enzyme activity, (ii) is allosterically inhibited by lysine, and (iii) adopts the canonical homo‐tetrameric structure in both solution and the crystal state. This study describes for the first time the structure, function and allostery of the bona fide DHDPS from A. tumefaciens, which offers insight into the rational design of pesticide agents for combating Crown Gall disease. Proteins 2014; 82:1869–1883. © 2014 Wiley Periodicals, Inc.     

Kinetic evidence that the high-affinity glucose 6-phosphate site on hexokinase I Is the active site

Two mechanisms have been suggested to account for the regulation of brain hexokinase by glucose 6-phosphate. One mechanism places glucose-6-P at an allosteric site, remote from the active site, while the second describes glucose-6-P as a simple product inhibitor of the enzyme, binding at the γ phosphate subsite within the ATP locus of the active site. To distinguish between these possibilities, we have undertaken a study of the back reaction of hexokinase I. Our data indicate that glucose-6-P displays classical Michaelis-Menten kinetics with brain hexokinase. This finding is consistent only with the high-affinity glucose-6-P site on the enzyme being the catalytic site. The dissociation constant, estimated from the initial-rate experiments is approximately 25 μm, a value that agrees well with the inhibition constant for glucose-6-P in the forward direction. These findings are consistent with an earlier model (W. R. Ellison, J. D. Lueck and H. J. Fromm, (1975) J. Biol. Chem.250, 1864–1871), which maintains that glucose-6-P inhibition of brain hexokinase is a manifestation of product inhibition. In a recent paper, Lazo et al. (P. A. Lazo, A. Sols, and J. E. Wilson, (1980) J. Biol. Chem.255, 7548–7551) reported data obtained from binding studies with rat brain hexokinase at an elevated (250 μm) level of glucose-6-P. These authors believe that their results indicate multiple binding of glucose-6-P to the enzyme and interpret the data in terms of a high-affinity allosteric site and a low-affinity catalytic site. Our results are at variance with this interpretation and are consistent only with the high-affinity site for glucose-6-P on brain hexokinase being the active site.     

Structural and dynamic requirements for optimal activity of the essential bacterial enzyme dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS) is an essential enzyme involved in the lysine biosynthesis pathway. DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface. Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer evolved to optimise the dynamics within the tight-dimer. In order to gain insights into DHDPS flexibility and its relationship to quaternary structure and function, we performed comparative Molecular Dynamics simulation studies of native tetrameric and dimeric forms of DHDPS from E. coli and also the native dimeric form from methicillin-resistant Staphylococcus aureus (MRSA). These reveal a striking contrast between the dynamics of tetrameric and dimeric forms. Whereas the E. coli DHDPS tetramer is relatively rigid, both the E. coli and MRSA DHDPS dimers display high flexibility, resulting in monomer reorientation within the dimer and increased flexibility at the tight-dimer interface. The mutant E. coli DHDPS dimer exhibits disorder within its active site with deformation of critical catalytic residues and removal of key hydrogen bonds that render it inactive, whereas the similarly flexible MRSA DHDPS dimer maintains its catalytic geometry and is thus fully functional. Our data support the hypothesis that in both bacterial species optimal activity is achieved by fine tuning protein dynamics in different ways: E. coli DHDPS buttresses together two dimers, whereas MRSA dampens the motion using an extended tight-dimer interface.     

Biosynthesis of bacterial glycogen. Incorporation of pyridoxal phosphate into the allosteric activator site and an ADP-glucose-protected pyridoxal phosphate binding site of Escherichia coli B ADP-glucose synthase.

[3H]Pyridoxal-P can be covalently incorporated into Escherichia coli B mutant strain AC70R1 ADP-glucose synthase by reduction with NaBH4. Two distinct lysine residues can be modified by the allosteric activator pyridoxal-P. Incorporation of [3H]pyridoxal-P in the presence of substrate ADP-glucose + MgCl2 prevents pyridoxylation of an ADP-glucose-protected site and allows modification of the allosteric activator site. Incorporation of [3H]pyridoxal-P in the presence of allosteric effectors fructose-P2, 5'-AMP, or hexanediol-1,6-P2, protects against pyridoxylation of the allosteric activator site, and allows modification of the ADP-glucose-protected site. Incorporation of pyridoxal-P into the allosteric activator site results in modified enzyme of high activity form, even in the absence of fructose-P2. This modified enzyme, when assayed in the absence of fructose-P2, exhibits activation kinetics similar to nonpyridoxylated enzyme assayed in the presence of fructose-P2 and is still inhibited by 5'-AMP. These data suggest that the allosteric activator site of pyridoxylation is the fructose-P2 binding site, and is distinct from the inhibitor 5'-AMP binding site. Incorporation of pyridoxal-P into the ADP-glucose-protected site results in a decrease in enzyme activity. This pyridoxylated lysine could be involved with the binding of thesubstrates ADP-glucose, alpha-glucose-1-P, or PPi, or participate in the catalytic mechanism of the enzyme.     

Molecular evolution of an oligomeric biocatalyst functioning in lysine biosynthesis

Dihydrodipicolinate synthase (DHDPS) is critical to the production of lysine through the diaminopimelate (DAP) pathway. Elucidation of the function, regulation and structure of this key class I aldolase has been the focus of considerable study in recent years, given that the dapA gene encoding DHDPS has been found to be essential to bacteria and plants. Allosteric inhibition by lysine is observed for DHDPS from plants and some bacterial species, the latter requiring a histidine or glutamate at position 56 (Escherichia coli numbering) over a basic amino acid. Structurally, two DHDPS monomers form the active site, which binds pyruvate and (S)-aspartate β-semialdehyde, with most dimers further dimerising to form a tetrameric arrangement around a solvent-filled centre cavity. The architecture and behaviour of these dimer-of-dimers is explored in detail, including biophysical studies utilising analytical ultracentrifugation, small-angle X-ray scattering and macromolecular crystallography that show bacterial DHDPS tetramers adopt a head-to-head quaternary structure, compared to the back-to-back arrangement observed for plant DHDPS enzymes. Finally, the potential role of pyruvate in providing substrate-mediated stabilisation of DHDPS is considered.     

Exploring the allosteric mechanism of dihydrodipicolinate synthase by reverse engineering of the allosteric inhibitor binding sites and its application for lysine production

Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyzes the first committed reaction of l-lysine biosynthesis in bacteria and plants and is allosterically regulated by l-lysine. In previous studies, DHDPSs from different species were proved to have different sensitivity to l-lysine inhibition. In this study, we investigated the key determinants of feedback regulation between two industrially important DHDPSs, the l-lysine-sensitive DHDPS from Escherichia coli and l-lysine-insensitive DHDPS from Corynebacterium glutamicum, by sequence and structure comparisons and site-directed mutation. Feedback inhibition of E. coli DHDPS was successfully alleviated after substitution of the residues around the inhibitor’s binding sites with those of C. glutamicum DHDPS. Interestingly, mutagenesis of the lysine binding sites of C. glutamicum DHDPS according to E. coli DHDPS did not recover the expected feedback inhibition but an activation of DHDPS by l-lysine, probably due to differences in the allosteic signal transduction in the DHDPS of these two organisms. Overexpression of l-lysine-insensitive E. coli DHDPS mutants in E. coli MG1655 resulted in an improvement of l-lysine production yield by 46 %.     

Exploring the Structural Mechanism of Covalently Bound E3 Ubiquitin Ligase: Catalytic or Allosteric Inhibition?

Covalent inhibition has recently gained a resurgence of interest in several drug discovery areas. The expansion of this approach is based on evidence elucidating the selectivity and potency of covalent inhibitors when bound to particular amino acids of a biological target. The Nedd4-1, an E3 ubiquitin ligase, is characterized by two covalent binding sites, of which catalytic Cys^cat and allosteric Cys^allo are enclosed. This enzyme has demonstrated inhibition at both the above-mentioned binding sites; however, a detailed molecular understanding of the structural mechanism of inhibition upon Cys^cat and Cys^allo binding remains vague. This prompted us to provide the first account of investigating the preferential covalent binding mode and the underlying structural and molecular dynamic implications. Based on the molecular dynamic analyses, it was evident that although both catalytic and allosteric covalent binding led to greater stability of the enzyme, a preferential covalent mechanism of inhibition was seen in the allosteric-targeted system. This was supported by a more favorable binding energy in the allosteric site compared to the catalytic site, in addition to the larger number of residue interactions and stabilizing hydrogen bonds occurring in the allosteric covalent bound complex. The fundamental dynamic analysis presented in this report compliments, as well as adds to previous experimental findings, thus leading to a crucial understanding of the structural mechanism by which Nedd4-1 is inhibited. The findings from this study may assist in the design of more target-specific Nedd4-1 covalent inhibitors exploring the surface-exposed cysteine residues.     

Exploring a potential allosteric inhibition mechanism in the motor domain of human Eg-5

Kinesin-5 (Eg-5), microtubule motor protein, is one of the emerging drug targets in cancer research. Several inhibitors have been reported to bind the hEg-5 “motor domain” in two different locations that are potentially allosteric. Interestingly, the crystal structure of Eg-5 bound to benzimidazole unveils two chemically different allosteric pockets (PDB ID: 3ZCW). The allosteric modulators inhibit Eg-5 activity by causing conformational changes that affect nucleotide turnover rate. In the present work, three allosteric inhibitors were simulated along with the substrate nucleotides (ADP and ATP) to capture conformation changes induced by the allosteric inhibitors. To analyze the allosteric inhibition mechanism, we used dynamics cross-correlation, principal component analysis (PCA), and enthalpic calculations. The loop L5 interaction is determined by the type of substrate bind at the nucleotide binding site. The SW-II flexibility increased upon dual allosteric inhibition by SB-743921 and 6a. The ionic interaction between R221-E116 is observed only in the presence of two allosteric inhibitors. Also, we noticed that the α2/α3 helical orientation is responsible for the SW-1 loop position and substrate binding. Our simulation data suggest the critical chemical features required to block the motor domain by the allosteric inhibitors. The results summarized in this work will help the researchers to design better therapeutic agents targeting hEg-5.

Communicated by Ramaswamy H. Sarma     

New insights into the mechanism of dihydrodipicolinate synthase using isothermal titration calorimetry

Thermodynamic binding information, obtained via isothermal titration calorimetry (ITC), provides new insights into the binding of substrates, and of allosteric inhibitor interactions of dihydrodipicolinate synthase (DHDPS) from Escherichia coli. DHDPS catalyses the first committed step in (S)-lysine biosynthesis: the Schiff-base mediated aldol condensation of pyruvate with (S)-aspartate semi-aldehyde. Binding studies indicate that pyruvate is a weak binder (0.023 mM) but that (S)-ASA does not interact with the enzyme in the absence of a Schiff-base with pyruvate. These results support the assignment of a ping pong catalytic mechanism in which enthalpically driven Schiff-base formation (ΔH = −44.5 ± 0.1 kJ mol⁻¹) provides the thermodynamic impetus for pyruvate association. The second substrate, (S)-ASA, was observed to bind to a Schiff-base mimic (ΔH = −2.8 ± 0.1 kJ mol⁻¹) formed through the reduction of the intermediate pyruvyl–Schiff-base complex.