Mechanism of specific target recognition and RNA hydrolysis by ribonucleolytic toxin restrictocin

Restrictocin, a member of the fungal ribotoxin family, specifically cleaves a single phosphodiester bond in the 28S rRNA and potently inhibits eukaryotic protein synthesis. Residues Tyr47, His49, Glu95, Phe96, Pro97, Arg120, and His136 have been predicted to form the active site of restrictocin. In this study, we have individually mutated these amino acids to alanine to probe their role in restrictocin structure and function. The role of Tyr47, His49, Arg120, and His136 was further investigated by making additional mutants. Mutating Arg120 or His136 to alanine or the other amino acids rendered the toxin completely inactive, whereas mutating Glu95 to alanine only partially inactivated the toxin. Mutation of Phe96 and Pro97 to Ala had no effect on the activity of restrictocin. The Tyr47 to alanine mutant was inactive in inhibiting protein synthesis, and had a nonspecific ribonuclease activity on 28S rRNA similar to that shown previously for the His49 to Ala mutant. Unlike the His136 to Ala mutant, the double mutants containing Tyr47 or His49 mutated to alanine along with His136 did not compete with restrictocin to cause a significant reduction in the extent of cleavage of 28S rRNA. In a model of restrictocin and a 29-mer RNA substrate complex, residues Tyr47, His49, Glu95, Arg120, and His136 were found to be near the cleavage site on RNA. It is proposed that in restrictocin Glu95 and His136 are directly involved in catalysis, Arg120 is involved in the stabilization of the enzyme-substrate complex, Tyr47 provides structural stability to the active site, and His49 determines the substrate specificity.     

Crystal structure of meso-2,3-butanediol dehydrogenase in a complex with NAD+ and inhibitor mercaptoethanol at 1.7 A resolution for understanding of chiral substrate recognition mechanisms

The crystal structure of a ternary complex of meso-2,3-butanediol dehydrogenase with NAD+ and a competitive inhibitor, mercaptoethanol, has been determined at 1.7 A resolution by means of molecular replacement and refined to a final R-factor of 0.194. The overall structure is similar to those of the other short chain dehydrogenase/reductase enzymes. The NAD+ binding site, and the positions of catalytic residues Ser139, Tyr152, and Lys156 are also conserved. The crystal structure revealed that mercaptoethanol bound specifically to meso-2,3-butanediol dehydrogenase. Two residues around the active site, Gln140 and Gly183, forming hydrogen bonds with the inhibitor, are important but not sufficient for distinguishing stereoisomerism of a chiral substrate.     

The energetic network of hotspot residues between Cdc25B phosphatase and its protein substrate

We have investigated the functional network of hotspot residues at the remote docking site of two cell cycle regulators, namely Cdc25B phosphatase and its native protein substrate Cdk2-pTpY/CycA. Specifically, we have studied the roles of energetically important residues (Arg488, Arg492, Tyr497 on Cdc25B and Asp206 and Asp210 on Cdk2-pTpY/CycA) by generating a diverse set of substitutions and performing double and triple mutant cycle analyses. This transient protein-protein interaction is particularly well-suited for this mutagenic approach because various control experiments ensure that the effect of each mutation is limited to the interaction of interest. We find binary coupling energies for ion pairs and hydrogen bonds ranging from 0.7 kcal/mol to 3.9 kcal/mol and ternary coupling energies of 1.9 kcal/mol and 2.8 kcal/mol. Overall our biochemical analyses are in good agreement with the docked structure of the complex and suggest the following roles for the individual hotspot residues on Cdc25B. The most important contributor, Arg492, forms a specific and tight bidentate interaction with Asp206 and a weaker interaction with Asp210 that cannot be replaced by a Lys. Although Tyr497 does not directly participate in this ionic network, it is important for buttressing Arg492 using both its hydrophobic (aromatic ring) and hydrophilic characteristics (hydrogen bonding). Arg488 participates less specifically in the electrostatic network with Asp206 and Asp210 of the protein substrate as it can partially be replaced by Lys. Our data provide insight how a cluster of residues in a docking site remote from the site of the chemical reaction can bring about efficient and specific substrate recognition.     

Crystal structure of tetrameric homoisocitrate dehydrogenase from an extreme thermophile, Thermus thermophilus: involvement of hydrophobic dimer-dimer interaction in extremely high thermotolerance

The crystal structure of homoisocitrate dehydrogenase involved in lysine biosynthesis from Thermus thermophilus (TtHICDH) was determined at 1.85-A resolution. Arg85, which was shown to be a determinant for substrate specificity in our previous study, is positioned close to the putative substrate binding site and interacts with Glu122. Glu122 is highly conserved in the equivalent position in the primary sequence of ICDH and archaeal 3-isopropylmalate dehydrogenase (IPMDH) but interacts with main- and side-chain atoms in the same domain in those paralogs. In addition, a conserved Tyr residue (Tyr125 in TtHICDH) which extends its side chain toward a substrate and thus has a catalytic function in the related beta-decarboxylating dehydrogenases, is flipped out of the substrate-binding site. These results suggest the possibility that the conformation of the region containing Glu122-Tyr125 is changed upon substrate binding in TtHICDH. The crystal structure of TtHICDH also reveals that the arm region is involved in tetramer formation via hydrophobic interactions and might be responsible for the high thermotolerance. Mutation of Val135, located in the dimer-dimer interface and involved in the hydrophobic interaction, to Met alters the enzyme to a dimer (probably due to steric perturbation) and markedly decreases the thermal inactivation temperature. Both the crystal structure and the mutation analysis indicate that tetramer formation is involved in the extremely high thermotolerance of TtHICDH.     

Invariant amino acids essential for decoding function of polypeptide release factor eRF1

In eukaryotic ribosome, the N domain of polypeptide release factor eRF1 is involved in decoding stop signals in mRNAs. However, structure of the decoding site remains obscure. Here, we specifically altered the stop codon recognition pattern of human eRF1 by point mutagenesis of the invariant Glu55 and Tyr125 residues in the N domain. The 3D structure of generated eRF1 mutants was not destabilized as demonstrated by calorimetric measurements and calculated free energy perturbations. In mutants, the UAG response was most profoundly and selectively affected. Surprisingly, Glu55Arg mutant completely retained its release activity. Substitution of the aromatic ring in position 125 reduced response toward all stop codons. This result demonstrates the critical importance of Tyr125 for maintenance of the intact structure of the eRF1 decoding site. The results also suggest that Tyr125 is implicated in recognition of the 3d stop codon position and probably forms an H-bond with Glu55. The data point to a pivotal role played by the YxCxxxF motif (positions 125–131) in purine discrimination of the stop codons. We speculate that eRF1 decoding site is formed by a 3D network of amino acids side chains.     

Refined crystal structure of the potato inhibitor complex of carboxypeptidase A at 2.5 Å resolution

The exopeptidase carboxypeptidase A forms a tight complex with a 39 residue inhibitor protein from potatoes. We have determined the crystal structure of this complex, and refined the atomic model to a crystallographic R-factor of 0.196 at 2.5 Å resolution. The structure of the inhibitor protein is organized around a core of disulfide bridges. No α-helices or β-sheets are present in this protein, although there is one turn of 3₁₀ helix. The four carboxy-terminal residues of the inhibitor protein bind in the active site groove of carboxypeptidase A, defining binding subsites S′₁, S₁, S₂ and S₃ on the enzyme. The carboxy-terminal glycine of the inhibitor is cleaved from the remainder of the inhibitor in the complex, and remains trapped in the back of the active site pocket. Interactions between the inhibitor and residues Tyr248 and Arg71 of carboxypeptidase A resemble possible features of binding stages for substrates both prior and subsequent to peptide bond hydrolysis. Not all of these interactions would be available to different types of ester substrates, however, which may be in part responsible for the observed kinetic differences in hydrolysis between peptides and various classes of esters. With the exception of residues involved in the binding of the inhibitor protein (such as Tyr248), the structure of carboxypeptidase A as determined in the inhibitor complex is quite similar to the structure of the unliganded enzyme (Lipscomb et al., 1968), which was solved from an unrelated crystal form.     

The role of factor XIa (FXIa) catalytic domain exosite residues in substrate catalysis and inhibition by the Kunitz protease inhibitor domain of protease nexin 2

To select residues in coagulation factor XIa (FXIa) potentially important for substrate and inhibitor interactions, we examined the crystal structure of the complex between the catalytic domain of FXIa and the Kunitz protease inhibitor (KPI) domain of a physiologically relevant FXIa inhibitor, protease nexin 2 (PN2). Six FXIa catalytic domain residues (Glu(98), Tyr(143), Ile(151), Arg(3704), Lys(192), and Tyr(5901)) were subjected to mutational analysis to investigate the molecular interactions between FXIa and the small synthetic substrate (S-2366), the macromolecular substrate (factor IX (FIX)) and inhibitor PN2KPI. Analysis of all six Ala mutants demonstrated normal K(m) values for S-2366 hydrolysis, indicating normal substrate binding compared with plasma FXIa; however, all except E98A and K192A had impaired values of k(cat) for S-2366 hydrolysis. All six Ala mutants displayed deficient k(cat) values for FIX hydrolysis, and all were inhibited by PN2KPI with normal values of K(i) except for K192A, and Y5901A, which displayed increased values of K(i). The integrity of the S1 binding site residue, Asp(189), utilizing p-aminobenzamidine, was intact for all FXIa mutants. Thus, whereas all six residues are essential for catalysis of the macromolecular substrate (FIX), only four (Tyr(143), Ile(151), Arg(3704), and Tyr(5901)) are important for S-2366 hydrolysis; Glu(98) and Lys(192) are essential for FIX but not S-2366 hydrolysis; and Lys(192) and Tyr(5901) are required for both inhibitor and macromolecular substrate interactions.     

Structure of aldehyde reductase in ternary complex with coenzyme and the potent 20alpha-hydroxysteroid dehydrogenase inhibitor 3,5-dichlorosalicylic acid: implications for inhibitor binding and selectivity

The structure of aldehyde reductase (ALR1) in ternary complex with the coenzyme NADPH and 3,5-dichlorosalicylic acid (DCL), a potent inhibitor of human 20α-hydroxysteroid dehydrogenase (AKR1C1), was determined at a resolution of 2.41 Å. The inhibitor formed a network of hydrogen bonds with the active site residues Trp22, Tyr50, His113, Trp114 and Arg312. Molecular modelling calculations together with inhibitory activity measurements indicated that DCL was a less potent inhibitor of ALR1 (256-fold) when compared to AKR1C1. In AKR1C1, the inhibitor formed a 10-fold stronger binding interaction with the catalytic residue (Tyr55), non-conserved hydrogen bonding interaction with His222, and additional van der Waals contacts with the non-conserved C-terminal residues Leu306, Leu308 and Phe311 that contribute to the inhibitor’s selectivity advantage for AKR1C1 over ALR1.     

Crystallographic study of mechanism of ribonuclease T1-catalysed specific RNA hydrolysis

Ribonuclease T1 (RNase T1) cleaves the phosphodiester bond of RNA specifically at the 3'-end of guanosine. 2'-guanosinemonophosphate (2'-GMP) acts as inhibitor for this reaction and was cocrystallized with RNase T1. X-Ray analysis provided insight in the geometry of the active site and in the parts of the enzyme involved in the recognition of guanosine. RNase T1 is globular in shape and consists of a 4.5 turns alpha-helix lying "below" a four-stranded antiparallel beta-sheet containing recognition center as well as active site. The latter is indicated by the position of phosphate and sugar residues of 2'-GMP and shows that Glu58, His92 and Arg77 are active in phosphodiester hydrolysis. Guanine is recognized by a stretch of protein from Tyr42 to Tyr45. Residues involved in recognition are peptide NH and C = O, guanine O6 and N1H which form hydrogen bonds and a stacking interaction of Tyr45 on guanine. Although, on a theoretical basis, many specific amino acid-guanine interactions are possible, none is employed in the RNase T1.guanine recognition.     

Sequential side-chain residue motions transform the binary into the ternary state of DNA polymerase lambda

The nature of conformational transitions in DNA polymerase lambda (pol lambda), a low-fidelity DNA repair enzyme in the X-family that fills short nucleotide gaps, is investigated. Specifically, to determine whether pol lambda has an induced-fit mechanism and open-to-closed transition before chemistry, we analyze a series of molecular dynamics simulations from both the binary and ternary states before chemistry, with and without the incoming nucleotide, with and without the catalytic Mg(2+) ion in the active site, and with alterations in active site residues Ile(492) and Arg(517). Though flips occurred for several side-chain residues (Ile(492), Tyr(505), Phe(506)) in the active site toward the binary (inactive) conformation and partial DNA motion toward the binary position occurred without the incoming nucleotide, large-scale subdomain motions were not observed in any trajectory from the ternary complex regardless of the presence of the catalytic ion. Simulations from the binary state with incoming nucleotide exhibit more thumb subdomain motion, particularly in the loop containing beta-strand 8 in the thumb, but closing occurred only in the Ile(492)Ala mutant trajectory started from the binary state with incoming nucleotide and both ions. Further connections between active site residues and the DNA position are also revealed through our Ile(492)Ala and Arg(517)Ala mutant studies. Our combined studies suggest that while pol lambda does not demonstrate large-scale subdomain movements as DNA polymerase beta (pol beta), significant DNA motion exists, and there are sequential subtle side chain and other motions-associated with Arg(514), Arg(517), Ile(492), Phe(506), Tyr(505), the DNA, and again Arg(514) and Arg(517)-all coupled to active site divalent ions and the DNA motion. Collectively, these motions transform pol lambda to the chemistry-competent state. Significantly, analogs of these residues in pol beta (Lys(280), Arg(283), Arg(258), Phe(272), and Tyr(271), respectively) have demonstrated roles in determining enzyme efficiency and fidelity. As proposed for pol beta, motions of these residues may serve as gate-keepers by controlling the evolution of the reaction pathway before the chemical reaction.     

Structural insights into the human and avian IMP cyclohydrolase mechanism via crystal structures with the bound XMP inhibitor

Within de novo purine biosynthesis, the AICAR transformylase and IMP cyclohydrolase activities of the bifunctional enzyme ATIC convert the intermediate AICAR to the final product of the pathway, IMP. Identification of the AICAR transformylase active site and a proposed formyl transfer mechanism have already resulted from analysis of crystal structures of avian ATIC in complex with substrate and/or inhibitors. Herein, we focus on the IMPCH active site and the cyclohydrolase mechanism through comparison of crystal structures of XMP inhibitor complexes of human ATIC at 1.9 A resolution with the previously determined avian enzyme. This first human ATIC structure was also determined to ascertain whether any subtle structural differences, compared to the homologous avian enzyme, should be taken into account for structure-based inhibitor design. These structural comparisons, as well as comparative analyses with other IMP and XMP binding proteins, have enabled a catalytic mechanism to be formulated. The primary role of the IMPCH active site appears to be to induce a reconfiguration of the substrate FAICAR to a less energetically favorable, but more reactive, conformer. Backbone (Arg64 and Lys66) and side chain interactions (Thr67) in the IMPCH active site reorient the 4-carboxamide from the preferred conformer that binds to the AICAR Tfase active site to one that promotes intramolecular cyclization. Other backbone amides (Ile126 and Gly127) create an oxyanion hole that helps orient the formyl group for nucleophilic attack by the 4-carboxamide amine and then stabilize the anionic intermediate. Several other residues, including Lys66, Tyr104, Asp125, and Lys137', provide substrate specificity and likely enhance the catalytic rate through contributions to acid-base catalysis.     

Structures of three inhibitor complexes provide insight into the reaction mechanism of the human methylenetetrahydrofolate dehydrogenase/cyclohydrolase

Enzymes involved in tetrahydrofolate metabolism are of particular pharmaceutical interest, as their function is crucial for amino acid and DNA biosynthesis. The crystal structure of the human cytosolic methylenetetrahydrofolate dehydrogenase/cyclohydrolase (DC301) domain of a trifunctional enzyme has been determined previously with a bound NADP cofactor. While the substrate binding site was identified to be localized in a deep and rather hydrophobic cleft at the interface between two protein domains, the unambiguous assignment of catalytic residues was not possible. We succeeded in determining the crystal structures of three ternary DC301/NADP/inhibitor complexes. Investigation of these structures followed by site-directed mutagenesis studies allowed identification of the amino acids involved in catalysis by both enzyme activities. The inhibitors bind close to Lys56 and Tyr52, residues of a strictly conserved motif for active sites in dehydrogenases. While Lys56 is in a good position for chemical interaction with the substrate analogue, Tyr52 was found stacking against the inhibitors' aromatic rings and hence seems to be more important for proper positioning of the ligand than for catalysis. Also, Ser49 and/or Cys147 were found to possibly act as an activator for water in the cyclohydrolase step. These and the other residues (Gln100 and Asp125), with which contacts are made, are strictly conserved in THF dehydrogenases. On the basis of structural and mutagenesis data, we propose a reaction mechanism for both activities, the dehydrogenase and the cyclohydrolase.     

A structural factor responsible for substrate recognition by Bacillus sp. GL1 xanthan lyase that acts specifically on pyruvated side chains of xanthan

Xanthan is a bacterial heteropolysaccharide composed of pentasaccharide repeating units, i.e., a cellobiose as a backbone and a trisaccharide consisting of two mannoses and one glucuronic acid as a side chain. Nonreducing terminal mannose residues of xanthan side chains are partially pyruvated. Bacillus sp. GL1 xanthan lyase, a member of polysaccharide lyase family 8, acts specifically on pyruvated side chains of xanthan and yields pyruvated mannose through a beta-elimination reaction by using a single Tyr255 residue as base and acid catalysts. Here we show structural factors for substrate recognition by xanthan lyase through X-ray crystallographic and mutational analyses. The enzyme accommodates mannose and pyruvated mannose at the -1 subsite, although both inhibitor and dissociation constants of the two monosaccharides indicated that the affinity of pyruvated mannose for xanthan lyase is much higher than that of mannose. The high affinity of pyruvated mannose is probably due to the formation of additional hydrogen bonds between the carboxyl group of pyruvated mannose and amino acid residues of Tyr315 and Arg612. Site-directed mutagenesis of the two residues demonstrated that Arg612 is a key residue in recognizing pyruvated mannose. Arg612 is located in the protruding loop covering the substrate, suggesting that the loop functions as a lid that is responsible for the proper accommodation of the substrate at the active site.     

The catalytic mechanism of Drosophila alcohol dehydrogenase: evidence for a proton relay modulated by the coupled ionization of the active site Lysine/Tyrosine pair and a NAD+ ribose OH switch

The ionization properties of the active site residues in Drosophila lebanonensis alcohol dehydrogenase (DADH) were investigated theoretically by using an approach developed to account for multiple locations of the hydrogen atoms of the titratable and polar groups. The electrostatic calculations show that (a) the protonation/deprotonation transition of the binary complex of DADH is related to the coupled ionization of Tyr151 and Lys155 in the active site and (b) the pH dependence of the proton abstraction is correlated with a reorganization of the hydrogen bond network in the active site. On this basis, a proton relay mechanism for substrate dehydrogenation is proposed in which the O2' ribose hydroxyl group from the coenzyme has a key role and acts as a switch. The proton relay chain includes the active site catalytic residues, as well as a chain of eight water molecules that connects the active site with the bulk solvent.     

Crystal structure of Escherichia coli malate dehydrogenase. A complex of the apoenzyme and citrate at 1.87 A resolution.

The crystal structure of malate dehydrogenase from Escherichia coli has been determined with a resulting R-factor of 0.187 for X-ray data from 8.0 to 1.87 A. Molecular replacement, using the partially refined structure of porcine mitochondrial malate dehydrogenase as a probe, provided initial phases. The structure of this prokaryotic enzyme is closely homologous with the mitochondrial enzyme but somewhat less similar to cytosolic malate dehydrogenase from eukaryotes. However, all three enzymes are dimeric and form the subunit-subunit interface through similar surface regions. A citrate ion, found in the active site, helps define the residues involved in substrate binding and catalysis. Two arginine residues, R81 and R153, interacting with the citrate are believed to confer substrate specificity. The hydroxyl of the citrate is hydrogen-bonded to a histidine, H177, and similar interactions could be assigned to a bound malate or oxaloacetate. Histidine 177 is also hydrogen-bonded to an aspartate, D150, to form a classic His.Asp pair. Studies of the active site cavity indicate that the bound citrate would occupy part of the site needed for the coenzyme. In a model building study, the cofactor, NAD, was placed into the coenzyme site which exists when the citrate was converted to malate and crystallographic water molecules removed. This hypothetical model of a ternary complex was energy minimized for comparison with the structure of the binary complex of porcine cytosolic malate dehydrogenase. Many residues involved in cofactor binding in the minimized E. coli malate dehydrogenase structure are homologous to coenzyme binding residues in cytosolic malate dehydrogenase. In the energy minimized structure of the ternary complex, the C-4 atom of NAD is in van der Waals' contact with the C-3 atom of the malate. A catalytic cycle involves hydride transfer between these two atoms.     

Characterization of homoisocitrate dehydrogenase involved in lysine biosynthesis of an extremely thermophilic bacterium,Thermus thermophilus HB27, and evolutionary implication of beta-decarboxylating dehydrogenase

Although the presence of an enzyme that catalyzes beta-decarboxylating dehydrogenation of homoisocitrate to synthesize 2-oxoadipate has been postulated in the lysine biosynthesis pathway through alpha-aminoadipate (AAA), the enzyme has not yet been analyzed at all, because no gene encoding the enzyme has been identified until recently. A gene encoding a protein with a significant amino acid sequence identity to both isocitrate dehydrogenase and 3-isopropylmalate dehydrogenase was cloned from Thermus thermophilus HB27. The gene product produced in recombinant Escherichia coli cells demonstrated homoisocitrate dehydrogenase (HICDH) activity. A knockout mutant of the gene showed an AAA-auxotrophic phenotype, indicating that the gene product is involved in lysine biosynthesis through AAA. We therefore named this gene hicdh. HICDH, the gene product, did not catalyze the conversion of 3-isopropylmalate to 2-oxoisocaproate, a leucine biosynthetic reaction, but it did recognize isocitrate, a related compound in the tricarboxylic acid cycle, as well as homoisocitrate as a substrate. It is of interest that HICDH catalyzes the reaction with isocitrate about 20 times more efficiently than the reaction with the putative native substrate, homoisocitrate. The broad specificity and possible dual function suggest that this enzyme represents a key link in the evolution of the pathways utilizing citrate derivatives. Site-directed mutagenesis study reveals that replacement of Arg(85) with Val in HICDH causes complete loss of activity with isocitrate but significant activity with 3-isopropylmalate and retains activity with homoisocitrate. These results indicate that Arg(85) is a key residue for both substrate specificity and evolution of beta-decarboxylating dehydrogenases.     

Structural and mechanistic investigation of 3-deoxy-D-manno-octulosonate-8-phosphate synthase by solid-state REDOR NMR

15N?(31)P? REDOR NMR experiments were applied to lyophilized binary complexes of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS), with each of its natural substrates, phosphoenolpyruvate (PEP) and arabinose-5-phsophate (A5P), and with a mechanism-based inhibitor (K(i) = 0.4 microM), directly characterizing the active site basic residues involved in the binding of their carboxylate and phosphate moieties. KDO8PS was labeled uniformly with (15)N or [eta-(15)N(2)]Arg, and the ligands were selectively labeled with (13)C and (15)N. The NMR data established that PEP is bound by KDO8PS via a preserved set of structurally rigid and chemically unique Arg and Lys residues, with 5 A (upper limit) between epsilon-(15)N of this Lys and (31)P of PEP. A5P is bound in its cyclic forms to KDO8PS via a different set of Lys and Arg residues. The two sets arise from adjacent subsites that are capable of independent and sufficiently strong binding. The inhibitor is best characterized as an A5P-based substrate analogue inhibitor of KDO8PS. Five mutants in which highly conserved arginines were replaced with alanines were prepared and kinetically characterized. Our solid-state NMR observations complement the crystallographic structure of KDO8PS, and in combination with the mutagenesis results enable tentative assignment of the NMR-identified active site residues. Lys-138 and Arg-168 located at the most recessed part of the active site cavity are the chemically distinct and structurally rigid residues that bind PEP phosphate; R168A resulted in 0.1% of wild-type activity. Arg-63, exposed at the opening of the active site barrel, is the flexible residue with a generic chemical shift that binds A5P; R63A resulted in complete deactivation. The mechanistic implications of our results are discussed.     

Coagulation factor IXa: the relaxed conformation of Tyr99 blocks substrate binding.

BACKGROUND: Among the S1 family of serine proteinases, the blood coagulation factor IXa (fIXa) is uniquely inefficient against synthetic peptide substrates. Mutagenesis studies show that a loop of residues at the S2-S4 substrate-binding cleft (the 99-loop) contributes to the low efficiency. The crystal structure of porcine fIXa in complex with the inhibitor D-Phe-Pro-Arg-chloromethylketone (PPACK) was unable to directly clarify the role of the 99-loop, as the doubly covalent inhibitor induced an active conformation of fIXa. RESULTS: The crystal structure of a recombinant two-domain construct of human fIXa in complex with p-aminobenzamidine shows that the Tyr99 sidechain adopts an atypical conformation in the absence of substrate interactions. In this conformation, the hydroxyl group occupies the volume corresponding to the mainchain of a canonically bound substrate P2 residue. To accommodate substrate binding, Tyr99 must adopt a higher energy conformation that creates the S2 pocket and restricts the S4 pocket, as in fIXa-PPACK. The energy cost may contribute significantly to the poor K(M) values of fIXa for chromogenic substrates. In homologs, such as factor Xa and tissue plasminogen activator, the different conformation of the 99-loop leaves Tyr99 in low-energy conformations in both bound and unbound states. CONCLUSIONS: Molecular recognition of substrates by fIXa seems to be determined by the action of the 99-loop on Tyr99. This is in contrast to other coagulation enzymes where, in general, the chemical nature of residue 99 determines molecular recognition in S2 and S3-S4. This dominant role on substrate interaction suggests that the 99-loop may be rearranged in the physiological fX activation complex of fIXa, fVIIIa, and fX.     

The structure and mechanism of the type II dehydroquinase from Streptomyces coelicolor

The structure of the type II DHQase from Streptomyces coelicolor has been solved and refined to high resolution in complexes with a number of ligands, including dehydroshikimate and a rationally designed transition state analogue, 2,3-anhydro-quinic acid. These structures define the active site of the enzyme and the role of key amino acid residues and provide snap shots of the catalytic cycle. The resolution of the flexible lid domain (residues 21-31) shows that the invariant residues Arg23 and Tyr28 close over the active site cleft. The tyrosine acts as the base in the initial proton abstraction, and evidence is provided that the reaction proceeds via an enol intermediate. The active site of the structure of DHQase in complex with the transition state analog also includes molecules of tartrate and glycerol, which provide a basis for further inhibitor design.