Identification Of Active Site Residues Research Articles

Kinetic Characterization and Identification of Key Active Site Residues of the L-aspartate N-hydroxylase, CreE.

CreE is a flavin-dependent monooxygenase (FMO) that catalyzes three sequential nitrogen oxidation reactions of L-aspartate to produce nitrosuccinate, contributing to the biosynthesis of the antimicrobial and antiproliferative nautral product, cremeomycin. This compound contains a highly reactive diazo functional group in which the reaction of CreE is essential to its formation. Nitro and diazo functional groups can serve as potent electrophiles, important in some challenging nucleophilic addition reactions. Formation of these reactive groups positions CreE as a promising candidate for biomedical and synthetic applications. Here, we present the catalytic mechanism of CreE and the identification of active site residues critical to binding L-aspartate, aiding in future enzyme engineering efforts. Steady-state analysis demonstrated that CreE is very specific for NADPH over NADH and performs a highly coupled reaction with L-aspartate. Analysis of the rapid-reaction kinetics showed that flavin reduction is very fast, along with the formation of the oxygenating species, the C4a-hydroperoxyflavin. The slowest step observed was the dehydration of the flavin. Structural analysis and site-directed mutagenesis implicated T65, R291, and R440 in the binding L-aspartate. The data presented describes the catalytic mechanism and the active site architecture of this unique FMO.

Structural Evaluation and Conformational Dynamics of ZNF141T474I Mutation Provoking Postaxial Polydactyly Type A.

This study delineates the pathogenic association, miss-fold aggregation, and conformational paradigm of a missense variant (c.1420C > T; p.T474I) in ZNF141 gene segregating PAP through a molecular dynamics simulations approach. In ZNF141 protein, helices play a crucial role by attaching three specific target DNA base pairs. In ZNF141T474I protein, H1, H3, and H6 helices attain more flexibility by acquiring loop conformation. The outward disposition of the proximal portion of H9-helix in mutant protein occurs due to the loss of prior beta-hairpins at the C terminal region of the C2-H2 domain. The loss of hydrogen bonds and exposure of hydrophobic residues to solvent and helices turning to loops cause dysfunction of ZNF141 protein. These significant changes in the stability and conformation of the mutant protein were validated using essential dynamics and cross-correlation maps, which revealed that upon point mutation, the overall motion of the proteins and the correlation between them were completely different, resulting in Postaxial polydactyly type A. This study provides molecular insights into the structural association of ZNF141 protein with PAP type A. Identification of active site residues and legends offers new therapeutic targets for ZNF141 protein. Further, it reiterates the functional importance of the last residue of a protein.

Identification of Active Site Residues of the Siderophore Synthesis Enzyme PvdF and Evidence for Interaction of PvdF with a Substrate-Providing Enzyme.

The problematic opportunistic pathogen Pseudomonas aeruginosa secretes a siderophore, pyoverdine. Pyoverdine scavenges iron needed by the bacteria for growth and for pathogenicity in a range of different infection models. PvdF, a hydroxyornithine transformylase enzyme, is essential for pyoverdine synthesis, catalysing synthesis of formylhydroxyornithine (fOHOrn) that forms part of the pyoverdine molecule and provides iron-chelating hydroxamate ligands. Using a mass spectrometry assay, we confirm that purified PvdF catalyses synthesis of fOHOrn from hydroxyornithine and formyltetrahydrofolate substrates. Site directed mutagenesis was carried out to investigate amino acid residues predicted to be required for enzymatic activity. Enzyme variants were assayed for activity in vitro and also in vivo, through measuring their ability to restore pyoverdine production to a pvdF mutant strain. Variants at two putative catalytic residues N168 and H170 greatly reduced enzymatic activity in vivo though did not abolish activity in vitro. Change of a third residue D229 abolished activity both in vivo and in vitro. A change predicted to block entry of N10-formyltetrahydrofolate (fTHF) to the active site also abolished activity both in vitro and in vivo. A co-purification assay showed that PvdF binds to an enzyme PvdA that catalyses synthesis of hydroxyornithine, with this interaction likely to increase the efficiency of fOHOrn synthesis. Our findings advance understanding of how P. aeruginosa synthesises pyoverdine, a key factor in host–pathogen interactions.

Computationally guided identification of Akt-3, a serine/threonine kinase inhibitors: Insights from homology modelling, structure-based screening, molecular dynamics and quantum mechanical calculations

Chemical entities targeting kinase signalling pathways serve as a potential strategy to combat malignancies. Protein Kinase B or Akt is a validated target for various malignancies and Akt3 remains the least explored isoform among all its isoforms. Initially, homology modelling technique was used for generating protein structure and further validation was performed using molecular dynamics simulation and Ramachandran plot. The validated protein structure was then subjected for active site analysis which led to identification of active site residues based on metrics provided by site score. The important residues in binding site were identified as Thr81, Asp271 and Asp289 for binding energetics and inhibition. Subsequently, virtual screening methodologies were used for identification of novel hits for inhibition of Protein Kinase B or Akt3. This led to the identification of two hits, i.e. thiophene derivative and thieno-pyridine derivative which were selected on the basis of their binding affinity and drug likeliness. These identified hits were subjected for molecular dynamics simulations, quantum mechanical and synthetic accessibility studies. The role of crucial residues in binding site stood validated as suggested by molecular dynamics simulations studies. Communicated by Ramaswamy H. Sarma

Identification of active site residues implies a two-step catalytic mechanism for acyl-ACP thioesterase

In plants and bacteria that use a Type II fatty acid synthase, isozymes of acyl-acyl carrier protein (ACP) thioesterase (TE) hydrolyze the thioester bond of acyl-ACPs, terminating the process of fatty acid biosynthesis. These TEs are therefore critical in determining the fatty acid profiles produced by these organisms. Past characterizations of a limited number of plant-sourced acyl-ACP TEs have suggested a thiol-based, papain-like catalytic mechanism, involving a triad of Cys, His, and Asn residues. In the present study, the sequence alignment of 1019 plant and bacterial acyl-ACP TEs revealed that the previously proposed Cys catalytic residue is not universally conserved and therefore may not be a catalytic residue. Systematic mutagenesis of this residue to either Ser or Ala in three plant acyl-ACP TEs, CvFatB1 and CvFatB2 from Cuphea viscosissima and CnFatB2 from Cocos nucifera, resulted in enzymatically active variants, demonstrating that this Cys residue (Cys348 in CvFatB2) is not catalytic. In contrast, the multiple sequence alignment, together with the structure modeling of CvFatB2, suggests that the highly conserved Asp309 and Glu347, in addition to previously proposed Asn311 and His313, may be involved in catalysis. The substantial loss of catalytic competence associated with site-directed mutants at these positions confirmed the involvement of these residues in catalysis. By comparing the structures of acyl-ACP TE and the Pseudomonas 4-hydroxybenzoyl-CoA TE, both of which fold in the same hotdog tertiary structure and catalyze the hydrolysis reaction of thioester bond, we have proposed a two-step catalytic mechanism for acyl-ACP TE that involves an enzyme-bound anhydride intermediate.

Identification of active site residues of Fenugreek β-amylase: Chemical modification and in silico approach

The amino acid sequence of Fenugreek β-amylase is not available in protein data bank. Therefore, an attempt has been made to identify the catalytic amino acid residues of enzyme by employing studies of pH dependence of enzyme catalysis, chemical modification and bioinformatics. Treatment of purified Fenugreek β-amylase with EDAC in presence of glycine methyl ester and sulfhydryl group specific reagents (IAA, NEM and p-CMB), followed a pseudo first-order kinetics and resulted in effective inactivation of enzyme. The reaction with EDAC in presence of NTEE (3-nitro-l-tyrosine ethylester) resulted into modification of two carboxyl groups per molecule of enzyme and presence of one accessible sulfhydryl group at the active site, per molecule of enzyme was ascertained by titration with DTNB. The above results were supported by the prevention of inactivation of enzyme in presence of substrate. Based on MALDI-TOF analysis of purified Fenugreek β-amylase and MASCOT search, β-amylase of Medicago sativa was found to be the best match. To further confirm the amino acid involved in catalysis, homology modelling of β-amylase of M. sativa was performed. The sequence alignment, superimposition of template and target models, along with study of interactions involved in docking of sucrose and maltose at the active site, led to identification of Glu187, Glu381 and Cys344 as active site residues.

Structural and Biochemical Studies of Human 4-hydroxy-2-oxoglutarate Aldolase: Implications for Hydroxyproline Metabolism in Primary Hyperoxaluria

Background4-hydroxy-2-oxoglutarate (HOG) aldolase is a unique enzyme in the hydroxyproline degradation pathway catalyzing the cleavage of HOG to pyruvate and glyoxylate. Mutations in this enzyme are believed to be associated with the excessive production of oxalate in primary hyperoxaluria type 3 (PH3), although no experimental data is available to support this hypothesis. Moreover, the identity, oligomeric state, enzymatic activity, and crystal structure of human HOGA have not been experimentally determined.Methodology/Principal FindingsIn this study human HOGA (hHOGA) was identified by mass spectrometry of the mitochondrial enzyme purified from bovine kidney. hHOGA performs a retro-aldol cleavage reaction reminiscent of the trimeric 2-keto-3-deoxy-6-phosphogluconate aldolases. Sequence comparisons, however, show that HOGA is related to the tetrameric, bacterial dihydrodipicolinate synthases, but the reaction direction is reversed. The 1.97 Å resolution crystal structure of hHOGA bound to pyruvate was determined and enabled the modeling of the HOG-Schiff base intermediate and the identification of active site residues. Kinetic analyses of site-directed mutants support the importance of Lys196 as the nucleophile, Tyr168 and Ser77 as components of a proton relay, and Asn78 and Ser198 as unique residues that facilitate substrate binding.Conclusions/SignificanceThe biochemical and structural data presented support that hHOGA utilizes a type I aldolase reaction mechanism, but employs novel residue interactions for substrate binding. A mapping of the PH3 mutations identifies potential rearrangements in either the active site or the tetrameric assembly that would likely cause a loss in activity. Altogether, these data establish a foundation to assess mutant forms of hHOGA and how their activity could be pharmacologically restored.

Crystal Structure Analysis of the Polysialic Acid Specific O-Acetyltransferase NeuO

The major virulence factor of the neuroinvasive pathogen Escherichia coli K1 is the K1 capsule composed of α2,8-linked polysialic acid (polySia). K1 strains harboring the CUS-3 prophage modify their capsular polysaccharide by phase-variable O-acetlyation, a step that is associated with increased virulence. Here we present the crystal structure of the prophage-encoded polysialate O-acetyltransferase NeuO. The homotrimeric enzyme belongs to the left-handed β-helix (LβH) family of acyltransferases and is characterized by an unusual funnel-shaped outline. Comparison with other members of the LβH family allowed the identification of active site residues and proposal of a catalytic mechanism and highlighted structural characteristics of polySia specific O-acetyltransferases. As a unique feature of NeuO, the enzymatic activity linearly increases with the length of the N-terminal poly-ψ-domain which is composed of a variable number of tandem copies of an RLKTQDS heptad. Since the poly-ψ-domain was not resolved in the crystal structure it is assumed to be unfolded in the apo-enyzme.

Probing the Basis of Domain-Dependent Inhibition Using Novel Ketone Inhibitors of Angiotensin-Converting Enzyme

Human angiotensin-converting enzyme (ACE) has two homologous domains, the N and C domains, with differing substrate preferences. X-ray crystal structures of the C and N domains complexed with various inhibitors have allowed identification of active site residues that might be important for the molecular basis of this selectivity. However, it is unclear to what extent the different residues contribute to substrate domain selectivity. Here, cocrystal structures of human testis ACE, equivalent to the C domain, have been determined with two novel C domain-selective ketomethylene inhibitors, (5 S)-5-[( N-benzoyl)amino]-4-oxo-6-phenylhexanoyl- l-tryptophan (kAW) and (5 S)-5-[( N-benzoyl)amino]-4-oxo-6-phenylhexanoyl- l-phenylalanine (kAF). The ketone groups of both inhibitors bind to the zinc ion as a hydrated geminal diolate, demonstrating the ability of the active site to catalyze the formation of the transition state. Moreover, active site residues involved in inhibitor binding have been mutated to their N domain counterparts, and the effect of the mutations on inhibitor binding has been determined. The C domain selectivity of these inhibitors was found to result from interactions between bulky hydrophobic side chain moieties and C domain-specific residues F391, V518, E376, and V380 (numbering of testis ACE). Mutation of these residues decreased the affinity for the inhibitors 4-20-fold. T282, V379, E403, D453, and S516 did not contribute individually to C domain-selective inhibitor binding. Further domain-selective inhibitor design should focus on increasing both the affinity and selectivity of the side chain moieties.

Nicotinamide riboside kinase structures reveal new pathways to NAD+.

The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD+) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD+ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD+.

Identification of active site residues of the inverting glycosyltransferase Cgs required for the synthesis of cyclic β-1,2-glucan, a Brucella abortus virulence factor

Brucella abortus cyclic glucan synthase (Cgs) is a 320-kDa (2868-amino acid) polytopic integral inner membrane protein responsible for the synthesis of the virulence factor cyclic beta-1,2-glucan by a novel mechanism in which the enzyme itself acts as a protein intermediate. Cgs functions as an inverting processive beta-1,2-autoglucosyltransferase and has the three enzymatic activities required for the synthesis of the cyclic glucan: initiation, elongation, and cyclization. To gain further insight into the protein domains that are essential for the enzymatic activity, we have compared the Cgs sequence with other glycosyltransferases (GTs). This procedure allowed us to identify in the Cgs region (475-818) the widely spaced D, DxD, E/D, (Q/R)xxRW motif that is highly conserved in the active site of numerous GTs. By site-directed mutagenesis and in vitro and in vivo activity assays, we have demonstrated that most of the amino acid residues of this motif are essential for Cgs activity. These sequence and site-directed mutagenesis analyses also indicate that Cgs should be considered a bi-functional modular GT, with an N-terminal GT domain belonging to a new GT family related to GT-2 (GT-84) followed by a GH-94 glycoside hydrolase C-terminal domain. Furthermore, over-expression of inactive mutants results in wild-type (WT) production of cyclic glucan when bacteria co-express the mutant and the WT form, indicating that Cgs may function in the membrane as a monomeric enzyme. Together, these results are compatible with a single addition model by which Cgs acts in the membrane as a monomer and uses the identified motif to form a single center for substrate binding and glycosyl-transfer reaction.

Structural Determinants for Inhibitor Specificity and Selectivity in PDE2A Using the Wheat Germ in Vitro Translation System

Phosphodiesterases (PDEs) modulate signaling by cyclic nucleotides in diverse processes such as cardiac contractility, platelet aggregation, lipolysis, glycogenolysis, and smooth muscle contraction. Cyclic guanosine monophosphate (cGMP) stimulated human phosphodiesterase 2 (PDE2) is expressed mainly in brain and heart tissues. PDE2A is involved in the regulation of blood pressure and fluid homeostasis by the atrial natriuretic peptide (ANP), making PDE2-type enzymes important targets for drug discovery. The design of more potent and selective inhibitors of PDE2A for the treatment of heart disease would be greatly aided by the identification of active site residues in PDE2A that determine substrate and inhibitor selectivity. The identification of active site residues through traditional mutational studies involves the time-consuming and tedious purification of a large number of mutant proteins from overexpressing cells. Here we report an alternative approach to rapidly produce active site mutants of human PDE2A and identify their enzymatic properties using a wheat germ in vitro translation (IVT, also known as cell-free translation) system. We also present the crystal structure of the catalytic domain of human PDE2A determined at 1.7 A resolution, which provided a framework for the rational design of active site mutants. Using a rapid IVT approach for expression of human PDE2A mutants, we identified the roles of active site residues Asp811, Gln812, Ile826, and Tyr827 in inhibitor and substrate selectivity for PDE2A.

Identification of active site residues in mevalonate diphosphate decarboxylase: implications for a family of phosphotransferases.

A combination of sequence homology analyses of mevalonate diphosphate decarboxylase (MDD) proteins and structural information for MDD leads to the hypothesis that Asp 302 and Lys 18 are active site residues in MDD. These residues were mutated to replace acidic/basic side chains and the mutant proteins were isolated and characterized. Binding and competitive displacement studies using trinitrophenyl-ATP, a fluorescent analog of substrate ATP, indicate that these mutant enzymes (D302A, D302N, K18M) retain the ability to stoichiometrically bind nucleotide triphosphates at the active site. These observations suggest the structural integrity of the mutant MDD proteins. The functional importance of mutated residues was evaluated by kinetic analysis. The 10(3) and 10(5)-fold decreases in k(cat) observed for the Asp 302 mutants (D302N and D302A, respectively) support assignment of a crucial catalytic role to Asp 302. A 30-fold decrease in activity and a 16-fold inflation of the K(m) for ATP is documented for the K18M mutant, indicating that Lys 18 influences the active site but is not crucial for reaction chemistry. Demonstration of the influence of conserved aspartate 302 appears to represent the first documentation of the functional importance of a residue in the MDD catalytic site and affords insight into phosphotransferase reactions catalyzed by a variety of enzymes in the galactokinase, homoserine kinase, mevalonate kinase, phosphom-evalonate kinase (GHMP kinase) family.

Identification of Active Site Residues of the Adeno-associated Virus Type 2 Rep Endonuclease

Adeno-associated virus type 2 Rep endonuclease activity is necessary for both viral DNA replication and site-specific integration of the viral genome into human chromosome 19. The biochemical activities required for site-specific endonuclease activity (namely specific DNA binding and transesterification activity) have been mapped to the amino-terminal domain of the AAV2 Rep protein. The amino-terminal 208 amino acids are alone sufficient for site-specific endonuclease activity, and nicking by this domain is metal-dependent. To identify this metal-binding site, we have employed a cysteine mutagenesis approach that targets conserved acidic amino acids. By using this technique, we provide functional biochemical data supporting a role for glutamate 83 in the coordination of metal ions in the context of Rep endonuclease activity. In addition, our biochemical data suggest that glutamate 164, although not involved in the coordination of metal ions, is closely associated with the active site. Thus, in lieu of a crystal structure for the AAV type 2 amino-terminal domain, our data corroborate the recently published structural studies of the AAV type 5 endonuclease and suggest that although the two enzymes are not highly conserved with respect to the AAV family, their active sites are highly conserved.

Identification of active site residues involved in metal cofactor binding and stereospecific substrate recognition in Mammalian tyrosinase. Implications to the catalytic cycle.

Tyrosinase (Tyr) and tyrosinase-related proteins (Tyrps) 1 and 2 are the enzymes responsible for mammalian melanogenesis. They display high similarity but different substrate and reaction specificities. Loss-of-function mutations lead to several forms of albinism or other pigmentation disorders. They share two conserved metal binding sites (CuA and CuB) which, in Tyr, bind copper. To define some structural determinants for these differences, we mutated Tyr at selected residues on the basis of (i) conservation of the original residues in most tyrosinases, (ii) their nonconservative substitution in the Tyrps, and (iii) their possible involvement as an endogenous bridge between the copper pair. Two mutations at the CuA site, S192A and E193Q, did not affect Tyr activities, thus excluding S192 and E193 as endogenous ligands of the copper pair. Concerning CuB, the H390Q mutation completely abolished Tyr activity, whereas Q378H and H389L mutants showed 10-20% residual specific activities. Their kinetic behavior suggests that (i) H390 is the actual third ligand for CuB, (ii) H389 is critical for stereospecific recognition of o-diphenols but not monophenols, and (iii) the involvement in metal binding of the central extra H residue at the Tyrps CuB site is unlikely. However, replacement of Q (in Tyr) by H (in Tyrps) greatly diminished the affinity for L-dopa, consistent with the low/null tyrosinase activity of the Tyrps. These are the first data showing a physical difference in docking of mono- and o-diphenols to the Tyr active site, and they are used to propose a revised scheme of the catalytic cycle.

Cloning, expression, purification and characterization of patatin, a novel phospholipase A.

Patatin is the major protein constituent of potato tubers and displays broad esterase activity. The native enzyme actually belongs to a highly homologous multigene family of vacuolar glycoproteins. From these, the patB2 patatin gene was selected and cloned into pUC19 without its signal sequence but with an N-terminal histidine-tag. This patatin was overexpressed under the control of the lac promotor in Escherichia coli strain DH5alpha. The protein was recovered as inclusion bodies, folded into its native state by solubilization in urea and purified to homogeneity. Starting with one gram of inclusion bodies, 19 mg of pure and active recombinant patatin was isolated, with even higher specific activity than the glycosylated wild-type patatin purified from potato tubers. The purified enzyme showed esterolytic activity with p-nitrophenylesters dissolved in Triton X-100 micelles. The activity of patatin on p-nitrophenylesters with different carbon chain lengths showed an optimum for p-nitrophenylesters with 10 carbon atoms. Besides general esterolytic activity, the pure enzyme was found to display high phospholipase A activity in particular with the substrates 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC(8)PCho) (127 U.mg(-1)) and 1,2-dinonanoyl-sn-glycero-3-phosphocholine (diC(9)PCho) (109 U.mg(-1)). Recently, the structure of human cytosolic PLA(2) (cPLA(2)) was solved, showing a novel Ser-Asp active site dyad [1]. Based on a partial sequence alignment of patatin with human cPLA(2), we propose that patatin contains a similar active site dyad. To verify this assumption, conserved Ser, Asp and His residues in the family of patatins have been modified in patatin B2. Identification of active site residues was based on the observation of correctly folded but inactive variants. This led to the assignment of Ser54 and Asp192 as the active site serine and aspartate residues in patatin B2, respectively.

Identification of Active Site Residues in Glucosylceramide Synthase: A NUCLEOTIDE-BINDING/CATALYTIC MOTIF CONSERVED WITH PROCESSIVE β-GLYCOSYLTRANSFERASES

Glucosylceramide synthase (GCS) transfers glucose from UDP-Glc to ceramide, catalyzing the first glycosylation step in the formation of higher order glycosphingolipids. The amino acid sequence of GCS was reported to be dissimilar from other proteins, with no identifiable functional domains. We previously identified His-193 of rat GCS as an important residue in UDP-Glc and GCS inhibitor binding; however, little else is known about the GCS active site. Here, we identify key residues of the GCS active site by performing biochemical and site-directed mutagenesis studies of rat GCS expressed in bacteria. First, we found that Cys-207 was the primary residue involved in GCS N-ethylmaleimide sensitivity. Next, we showed by multiple alignment that the region of GCS flanking His-193 and Cys-207 (amino acids 89-278) contains a D1,D2,D3,(Q/R)XXRW motif found in the putative active site of processive beta-glycosyltransferases (e.g. cellulose, chitin, and hyaluronan synthases). Site-directed mutagenesis studies demonstrated that most of the highly conserved residues were essential for GCS activity. We also note that GCS and processive beta-glycosyltransferases are topologically similar, possessing cytosolic active sites, with putative transmembrane domains immediately N-terminal to the conserved domain. These results provide the first extensive information on the GCS active site and show that GCS and processive beta-glycosyltransferases possess a conserved substrate-binding/catalytic domain.

Direct Identification of Enzyme Active Site Residues by Solid-State REDOR NMR: Application to KDO8P Synthase

In this communication we report the first direct identification of active site residues of the enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS). This is accomplished by the application of rotational-echo double-resonance1 (REDOR) solid-state NMR experiments to lyophilized binary complexes of uniformly 15N-labeled KDO8PS with each of its natural substrates, phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate (A5P). Its complex with the mechanism-based inhibitor, 2 (Scheme 1), the most potent inhibitor known to date2 (Ki ) 0.4 μM), is studied as well and its binding is compared to that of the natural substrates. The REDOR technique1 is based on selective reintroduction of (magnetic) dipolar interactions between pairs of specific types of nuclei, and in favorable cases enables their accurate measurement from which distance constraints can be obtained.3 Its application for the study of protein-ligand interactions is achieved by incorporation of stable isotopes at known positions in the protein that are proximate to (labeled) ligand moieties. In previous studies, REDOR measured distances were shown to be in excellent agreement with X-ray data.3a,b Moreover, 31P{13C} REDOR was employed to investigate the conformation of the bound inhibitor glyphosate in the enzyme-substrate-inhibitor ternary complex of 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS),3c for which no X-ray structure is available. REDOR, e.g., 31P{13C} and 15N {31P}, was also used for the determination of structural constraints in the above ternary complex,3d and to directly monitor transient enzyme-intermediate complexes of EPSPS3e and of UDP-GlcNac enolpyruvoyl transferase (Mur Z).3f The enzyme KDO8PS catalyzes the condensation reaction between A5P and PEP to form KDO8P and inorganic phosphate (Pi, Scheme 1).4 This important enzymatic reaction plays a crucial role in the assembly process of lipopolysaccharides of most Gramnegative bacteria,5 and is therefore an attractive target for the design of novel antibacterial drugs. Interestingly, unlike most PEPutilizing enzymes that cleave the high-energy P-O bond of PEP, KDO8PS and its family members (Mur Z, EPSPS, and 3-deoxyD-arabino-heptulosonate-7-phosphate synthase6 (DAHPS)) cleave the C-O bond of PEP.7 While the mechanisms of EPSPS8a (targeted by the herbicide glyphosate) and Mur Z8b (targeted by the antibiotic fosfomycin) were unambiguously characterized, the mechanisms of KDO8PS and DAHPS continue to be uncertain. Recent studies9a,2 on KDO8PS, including the synthesis and evaluation of the first acyclic bisubstrate inhibitor (2),2 supported a mechanism involving the formation of an acyclic bisphosphate intermediate 1 (Scheme 1).9b Mutagenesis studies10 identified in E. coli KDO8PS two vital cysteines, Cys38 and Cys166, yet their specific role still remains unclear. In addition, since to date neither the crystal structure nor the identity of the active site residues of KDO8PS are known, rational design of potent inhibitors for this enzyme is impaired. As part of our study on the structurefunction relationship of KDO8PS, [u-15N]KDO8PS was prepared and interactions between 15N enzyme labels to 31P nuclei of the ligands were examined by REDOR NMR.11 The 15N NMR CPMAS echo spectrum of [u-15N]KDO8PS binary complex with PEP (Figure 1a) shows resolved peaks for the nitrogen atoms of 10 side-chain arginine (guanidino: internal , 60 ppm; terminal η, 49 ppm), 22 lysine ( 9.0 ppm) residues, and an intense amide peak (95 ppm). Peaks of six side-chain histidines (δ1 224; 2 143 ppm) are not shown. In addition, detailed inspection of this spectrum reveals fine structure for the arginine (56.0 ppm), and a high field shoulder for lysine (3.0 ppm), attributed to a single arginine and to a single lysine residue residing in a distinct chemical environment. Interestingly, these two peaks are also present in the 15N CPMAS reference spectra of the other two complexes, KDO8PS-A5P and KDO8PS-2 (spectra not shown), implying that they arise from chemically distinct environments inherent to the enzyme itself, and are therefore not a result of interaction with a specific ligand. As will be shown below, these residues will be associated with specific recognition and binding of PEP by the enzyme.

Identification of active site residues in glycosidases by use of tandem mass spectrometry.

Identification of active site residues in glycosidases by use of tandem mass spectrometry.

1.68-Å Crystal Structure of Endopolygalacturonase II fromAspergillus niger and Identification of Active Site Residues by Site-directed Mutagenesis

Polygalacturonases specifically hydrolyze polygalacturonate, a major constituent of plant cell wall pectin. To understand the catalytic mechanism and substrate and product specificity of these enzymes, we have solved the x-ray structure of endopolygalacturonase II of Aspergillus niger and we have carried out site-directed mutagenesis studies. The enzyme folds into a right-handed parallel beta-helix with 10 complete turns. The beta-helix is composed of four parallel beta-sheets, and has one very small alpha-helix near the N terminus, which shields the enzyme's hydrophobic core. Loop regions form a cleft on the exterior of the beta-helix. Site-directed mutagenesis of Asp(180), Asp(201), Asp(202), His(223), Arg(256), and Lys(258), which are located in this cleft, results in a severe reduction of activity, demonstrating that these residues are important for substrate binding and/or catalysis. The juxtaposition of the catalytic residues differs from that normally encountered in inverting glycosyl hydrolases. A comparison of the endopolygalacturonase II active site with that of the P22 tailspike rhamnosidase suggests that Asp(180) and Asp(202) activate the attacking nucleophilic water molecule, while Asp(201) protonates the glycosidic oxygen of the scissile bond.