The Schiff base complex of yeast 5-aminolaevulinic acid dehydratase with laevulinic acid.

The X-ray structure of the complex formed between yeast 5-aminolaevulinic acid dehydratase (ALAD) and the inhibitor laevulinic acid has been determined at 2.15 A resolution. The inhibitor binds by forming a Schiff base link with one of the two invariant lysines at the catalytic center: Lys263. It is known that this lysine forms a Schiff base link with substrate bound at the enzyme's so-called P-site. The carboxyl group of laevulinic acid makes hydrogen bonds with the side-chain-OH groups of Tyr329 and Ser290, as well as with the main-chain >NH group of Ser290. The aliphatic moiety of the inhibitor makes hydrophobic interactions with surrounding aromatic residues in the protein including Phe219, which resides in the flap covering the active site. Our analysis strongly suggests that the same interactions will be made by P-side substrate and also indicates that the substrate that binds at the enzyme's A-site will interact with the enzyme's zinc ion bound by three cysteines (133, 135, and 143). Inhibitor binding caused a substantial ordering of the active site flap (residues 217-235), which was largely invisible in the native electron density map and indicates that this highly conserved yet flexible region has a specific role in substrate binding during catalysis.     

The X-ray structure of the plant like 5-aminolaevulinic acid dehydratase from Chlorobium vibrioforme complexed with the inhibitor laevulinic acid at 2.6 A resolution

5-Aminolaevulinic acid dehydratase (ALAD), an early enzyme of the tetrapyrrole biosynthesis pathway, catalyses the dimerisation of 5-aminolaevulinic acid to form the pyrrole, porphobilinogen. ALAD from Chlorobium vibrioforme is shown to form a homo-octameric structure with 422 symmetry in which each subunit adopts a TIM-barrel fold with a 30 residue N-terminal arm extension. Pairs of monomers associate with their arms wrapped around each other. Four of these dimers interact principally via their arm regions to form octamers in which each active site is located on the surface. The active site contains two invariant lysine residues (200 and 253), one of which (Lys253) forms a Schiff base link with the bound substrate analogue, laevulinic acid. The carboxyl group of the laevulinic acid forms hydrogen bonds with the side-chains of Ser279 and Tyr318. The structure was examined to determine the location of the putative active-site magnesium ion, however, no evidence for the metal ion was found in the electron density map. This is in agreement with previous kinetic studies that have shown that magnesium stimulates but is not required for activity. A different site close to the active site flap, in which a putative magnesium ion is coordinated by a glutamate carboxyl and five solvent molecules may account for the stimulatory properties of magnesium ions on the enzyme.     

Structure of aspartate racemase complexed with a dual substrate analogue, citric acid, and implications for the reaction mechanism

Pyrococcus horikoshii OT3 aspartate racemase (PhAspR) catalyzes the interconversion between L- and D-aspartate. The X-ray structure of PhAspR revealed a pseudo mirror-symmetric distribution of the residues around its active site, which is very reasonable for its chiral substrates, L-aspartate and D-aspartate. In this study, we have determined the crystal structure of an inactive mutant PhAspR complexed with a citric acid (Cit) at a resolution of 2.0 A. Cit contains the substrate analogue moieties of both L- and D-aspartate and exhibits a low competitive inhibition activity against PhAspR. In the structure, Cit binds to the catalytic site of PhAspR, which induced the conformational change to close the active site. The distance between the thiolates was estimated to be 7.4 A, representing a catalytic state and the substrate binding modes of PhAspR. Two conserved basic residues, Arg48 and Lys164, seem to be indispensable for PhAspR activity. Arg48 is thought to be responsible for recognizing carboxyl groups of the substrates L-/D-aspartates and stabilizing a reaction intermediate, and Lys164 is responsible for stabilizing a closed state structure. In this structure, the L-aspartate moiety of Cit is likely to take the substrate position of the PhAspR-substrate complex, which is very similar to that of Glutamate racemase. There is also another possibility that the two substrate analogue moieties of the bound Cit reflect the binding modes of both L- and D-aspartates. Based on the PhAspR-Cit complex structure, the reaction mechanism of aspartate racemase was elucidated.     

Histidine residues at the active site of maize δ-aminolevulinic acid dehydratase

Modification of maize δ-aminolevulinic acid dehydratase (ALAD) by diethylpyrocarbonate (DEP) caused rapid and complete inactivation of the enzyme. The inactivation showed saturation kinetics with a half inactivation time at saturating DEP equal to 0.3 min and K_DEP  0.3 mM. Substrate δ-aminolevulinic acid (ALA) and competitive inhibitor levulinic acid protected against inactivation, thereby indicating that DEP modifies the active site. The modified enzyme showed an increase in absorbance at 240 nm which was lost upon treatment with 0.8 M hydroxylamine. Most of the activity lost by DEP treatment could be restored after treatment with 0.8 M hydroxylamine. The results suggest that DEP modifies 7.4 residues/mole of the enzyme. These histidine residues are essential for catalysis by ALAD.     

The active site architecture of Pisum sativum beta-carbonic anhydrase is a mirror image of that of alpha-carbonic anhydrases

We have determined the structure of the beta-carbonic anhydrase from the dicotyledonous plant Pisum sativum at 1.93 A resolution, using a combination of multiple anomalous scattering off the active site zinc ion and non-crystallographic symmetry averaging. The mol- ecule assembles as an octamer with a novel dimer of dimers of dimers arrangement. Two distinct patterns of conservation of active site residues are observed, implying two potentially mechanistically distinct classes of beta-carbonic anhydrases. The active site is located at the interface between two monomers, with Cys160, His220 and Cys223 binding the catalytic zinc ion and residues Asp162 (oriented by Arg164), Gly224, Gln151, Val184, Phe179 and Tyr205 interacting with the substrate analogue, acetic acid. The substrate binding groups have a one to one correspondence with the functional groups in the alpha-carbonic anhydrase active site, with the corresponding residues being closely superimposable by a mirror plane. Therefore, despite differing folds, alpha- and beta-carbonic anhydrase have converged upon a very similar active site design and are likely to share a common mechanism.     

Crystal structures of salicylate 1,2-dioxygenase-substrates adducts: A step towards the comprehension of the structural basis for substrate selection in class III ring cleaving dioxygenases

The crystallographic structures of the adducts of salicylate 1,2-dioxygenase (SDO) with substrates salicylate, gentisate and 1-hydroxy-2-naphthoate, obtained under anaerobic conditions, have been solved and analyzed. This ring fission dioxygenase from the naphthalenesulfonate-degrading bacterium Pseudaminobacter salicylatoxidans BN12, is a homo-tetrameric class III ring-cleaving dioxygenase containing a catalytic Fe(II) ion coordinated by three histidine residues. SDO is markedly different from the known gentisate 1,2-dioxygenases or 1-hydroxy-2-naphthoate dioxygenases, belonging to the same class, because of its unique ability to oxidatively cleave salicylate, gentisate and 1-hydroxy-2-naphthoate. The crystal structures of the anaerobic complexes of the SDO reveal the mode of binding of the substrates into the active site and unveil the residues which are important for the correct positioning of the substrate molecules. Upon binding of the substrates the active site of SDO undergoes a series of conformational changes: in particular Arg127, His162, and Arg83 move to make hydrogen bond interactions with the carboxyl group of the substrate molecules. Unpredicted concerted displacements upon substrate binding are observed for the loops composed of residues 40-43, 75-85, and 192-198 where several aminoacidic residues, such as Leu42, Arg79, Arg83, and Asp194, contribute to the closing of the active site together with the amino-terminal tail (residues 2-15). Differences in substrate specificity are controlled by several residues located in the upper part of the substrate binding cavity like Met46, Ala85, Trp104, and Phe189, although we cannot exclude that the kinetic differences observed could also be generated by concerted conformational changes resulting from amino-acid mutations far from the active site.     

The x-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with substrate and three inhibitors.

The structures of 5-aminolaevulinic acid dehydratase (ALAD) complexed with substrate (5-aminolaevulinic acid) and three inhibitors: laevulinic acid, succinylacetone and 4-keto-5-aminolaevulinic acid, have been solved at high resolution. The ligands all bind by forming a covalent link with Lys263 at the active site. The structures define the interactions made by one of the two substrate moieties that bind to the enzyme during catalysis. All of the inhibitors induce a significant ordering of the flap covering the active site. Succinylacetone appears to be unique by inducing a number of conformational changes in loops covering the active site, which may be important for understanding the co-operative properties of ALAD enzymes. Succinylacetone is produced in large amounts by patients suffering from the hereditary disease type I tyrosinaemia and its potent inhibition of ALAD also has implications for the pathology of this disease. The most intriguing result is that obtained with 4-keto-5-amino-hexanoic acid, which seems to form a stable carbinolamine intermediate with Lys263. It appears that we have defined the structure of an intermediate of Schiff base formation that the substrate forms upon binding to the P-site of the enzyme.     

Evidence that both arginine 102 and arginine 747 are involved in substrate binding to neutral endopeptidase (EC 3.4.24.11)

Neutral endopeptidase (EC 3.4.24.11, NEP) is a Zn-metallopeptidase involved in the degradation of biologically active peptides, notably the enkephalins and atrial natriuretic peptide. Recently, the structure of the active site of this enzyme has been probed by site-directed mutagenesis, and 4 amino acid residues have been identified, namely 2 histidines (His583 and His587), which act as zinc-binding ligands, a glutamate (Glu584) involved in catalysis, and an arginine residue (Arg102), suggested to participate in substrate binding. Site-directed mutagenesis has now been used to investigate the role of 4 other arginine residues (Arg408, Arg409, Arg659, and Arg747) that have been proposed as possible active site residues and to further analyze the role of Arg102. In each case, the arginine was replaced with a methionine, and both enzymatic activity and the IC50 values of several NEP inhibitors were measured for the mutated enzymes and compared to wild-type enzyme. The results suggest that 2 arginines, Arg102 and Arg747, could both be important for substrate and inhibitor binding. Arg747 seems to be positioned to interact with the carbonyl amide group of the P'1 residue and can be modified when the enzyme is treated with the arginine-specific reagents phenylglyoxal and butanedione. Arg102 could be positioned to interact with the free carboxyl group of a P'2 residue in some substrates and inhibitors and can be modified by phenylglyoxal but not by butanedione. The results could explain the dual dipeptidylcarboxypeptidase and endopeptidase nature of NEP.     

Three-dimensional structure of porcine procarboxypeptidase B: a structural basis of its inactivity.

Procarboxypeptidase B is converted to enzymatically active carboxypeptidase B by limited proteolysis catalysed by trypsin, removing the long N-terminal activation segment of 95 amino acids. The three-dimensional crystal structure of procarboxypeptidase B from porcine pancreas has been determined at 2.3 A resolution and refined to a crystallographic R-factor of 0.169. The functional determinants of its enzymatic inactivity and of its activation by limited proteolysis have thus been unveiled. The activation segment folds in a globular region with an open sandwich antiparallel-alpha antiparallel-beta topology and in a C terminal alpha-helix which connects it to the enzyme moiety. The globular region (A7-A82) shields the preformed active site, and establishes specific interactions with residues important for substrate recognition. AspA41 forms a salt bridge with Arg145, which in active carboxypeptidase binds the C-terminal carboxyl group of substrate molecules. The connecting region occupies the putative extended substrate binding site. The scissile peptide bond cleaved by trypsin during activation is very exposed. Its cleavage leads to the release of the activation segment and to exposure of the substrate binding site. An open-sandwich folding has been observed in a number of other proteins and protein domains. One of them is the C-terminal fragment of L7/L12, a ribosomal protein from Escherichia coli that displays a topology similar to the activation domain of procarboxypeptidase.     

Structural basis for catalysis and substrate specificity of Agrobacterium radiobacter N-carbamoyl-D-amino acid amidohydrolase

N-Carbamoyl-d-amino acid amidohydrolase is an industrial biocatalyst to hydrolyze N-carbamoyl-d-amino acids for producing valuable d-amino acids. The crystal structure of N-carbamoyl-d-amino acid amidohydrolase in the unliganded form exhibits a alpha-beta-beta-alpha fold. To investigate the roles of Cys172, Asn173, Arg175, and Arg176 in catalysis, C172A, C172S, N173A, R175A, R176A, R175K, and R176K mutants were constructed and expressed, respectively. All mutants showed similar CD spectra and had hardly any detectable activity except for R173A that retained 5% of relative activity. N173A had a decreased value in kcat or Km, whereas R175K or R176K showed high Km and very low kcat values. Crystal structures of C172A and C172S in its free form and in complex form with a substrate, along with N173A and R175A, have been determined. Analysis of these structures shows that the overall structure maintains its four-layer architecture and that there is limited conformational change within the binding pocket except for R175A. In the substrate-bound structure, side chains of Glu47, Lys127, and C172S cluster together toward the carbamoyl moiety of the substrate, and those of Asn173, Arg175, and Arg176 interact with the carboxyl group. These results collectively suggest that a Cys172-Glu47-Lys127 catalytic triad is involved in the hydrolysis of the carbamoyl moiety and that Arg175 and Arg176 are crucial in binding to the carboxyl moiety, hence demonstrating substrate specificity. The common (Glu/Asp)-Lys-Cys triad observed among N-carbamoyl-d-amino acid amidohydrolase, NitFhit, and another carbamoylase suggests a conserved and robust platform during evolution, enabling it to catalyze the reactions toward a specific nitrile or amide efficiently.     

Role of two active site Glu residues in the molecular action of botulinum neurotoxin endopeptidase

Botulinum neurotoxin type A (BoNT/A) light chain (LC) is a zinc endopeptidase that causes neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. The X-ray crystal structure of the toxin reveals that His223 and His227 of the Zn(2+) binding motif HEXXH directly coordinate the active site zinc. Two Glu residues (Glu224 and Glu262) are also part of the active site, with Glu224 coordinating the zinc via a water molecule whereas Glu262 coordinates the zinc directly as the fourth ligand. In the past we have investigated the topographical role of Glu224 by replacing it with Asp thus reducing the side chain length by 1.4 A that reduced the endopeptidase activity dramatically [L. Li, T. Binz, H. Niemann, and B.R. Singh, Probing the role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain, Biochemistry 39 (2000) 2399-2405]. In this study we have moved the Glu 224 laterally by a residue (HXEXH) to assess its positional influence on the endopeptidase activity, which was completely lost. The functional implication of Glu262 was investigated by replacing this residue with aspartate and glutamine using site-directed mutagenesis. Substitution of Glu262 with Asp resulted in a 3-fold decrease in catalytic efficiency. This mutation did not induce any significant structural alterations in the active site and did not interfere with substrate binding. Substitution of Glu262 with Gln however, dramatically impaired the enzymatic activity and this is accompanied by global alterations in the active site conformation in terms of topography of aromatic amino acid residues, zinc binding, and substrate binding, resulting from the weakened interaction between the active site zinc and Gln. These results suggest a pivotal role of the negatively charged carboxyl group of Glu262 which may play a critical role in enhancing the stability of the active site with strong interaction with zinc. The zinc may thus play structural role in addition to its catalytic role.     

Probing the S1/S1' substrate binding pocket geometry of HIV-1 protease with modified aspartic acid analogues

Aspartates 25 and 125, the active site residues of HIV-1 protease, participate functionally in proteolysis by what is believed to be a general acid-general base mechanism. However, the structural role that these residues may play in the formation and maintenance of the neighboring S1/S1' substrate binding pockets remains largely unstudied. Because the active site aspartic acids are essential for catalysis, alteration of these residues to any other naturally occurring amino acid by conventional site-directed mutagenesis renders the protease inactive, and hence impossible to characterize functionally. To investigate whether Asp-25 and Asp-125 may also play a structural role that influences substrate processing, a series of active site protease mutants has been produced in a cell-free protein synthesizing system via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The suppressor tRNAs were activated with the unnatural aspartic acid analogues erythro-beta-methylaspartic acid, threo-beta-methylaspartic acid, or beta,beta-dimethylaspartic acid. On the basis of the specific activity measurements of the mutants that were produced, the introduction of the beta-methyl moiety was found to alter protease function to varying extents depending upon its orientation. While a beta-methyl group in the erythro orientation was the least deleterious to the specific activity of the protease, a beta-methyl group in the threo orientation, present in the modified proteins containing threo-beta-methylaspartate and beta,beta-dimethylaspartate, resulted in specific activities between 0 and 45% of that of the wild type depending upon the substrate and the substituted active site position. Titration studies of pH versus specific activity and inactivation studies, using an aspartyl protease specific suicide inhibitor, demonstrated that the mutant proteases maintained bell-shaped pH profiles, as well as suicide-inhibitor susceptibilities that are characteristic of aspartyl proteases. A molecular dynamics simulation of the beta-substituted aspartates in position 25 of HIV-1 protease indicated that the threo-beta-methyl moiety may partially obstruct the adjacent S1' binding pocket, and also cause reorganization within the pocket, especially with regard to residues Val-82 and Ile-84. This finding, in conjunction with the biochemical studies, suggests that the active site aspartate residues are in proximity to the S1/S1' binding pocket and may be spatially influenced by the residues presented in these pockets upon substrate binding. It thus seems possible that the catalytic residues cooperatively interact with the residues that constitute the S1/S1' binding pockets and can be repositioned during substrate binding to orient the active site carboxylates with respect to the scissile amide bond, a process that likely affects the facility of proteolysis.     

Roles of active site residues in Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase

In Pseudomonas aeruginosa, the dual-specificity enzyme phosphomannomutase/phosphoglucomutase catalyzes the transfer of a phosphoryl group from serine 108 to the hydroxyl group at the 1-position of the substrate, either mannose 6-P or glucose 6-P. The enzyme must then catalyze transfer of the phosphoryl group on the 6-position of the substrate back to the enzyme. Each phosphoryl transfer is expected to require general acid-base catalysis, provided by amino acid residues at the enzyme active site. An extensive survey of the active site residues by site-directed mutagenesis failed to identify a single key residue that mediates the proton transfers. Mutagenesis of active site residues Arg20, Lys118, Arg247, His308, and His329 to residues that do not contain ionizable groups produced proteins for which V(max) was reduced to 4-12% of that of the wild type. The fact that no single residue decreased catalytic activity more significantly, and that several residues had similar effects on V(max), suggested that the ensemble of active site amino acids act by creating positive electrostatic potential, which serves to depress the pK of the substrate hydroxyl group so that it binds in ionized form at the active site. In this way, the necessity of positioning the reactive hydroxyl group near a specific amino acid residue is avoided, which may explain how the enzyme is able to promote catalysis of both phosphoryl transfers, even though the 1- and 6-positions do not occupy precisely the same position when the substrate binds in the two different orientations in the active site. When Ser108 is mutated, the enzyme retains a surprising amount of activity, which has led to the suggestion that an alternative residue becomes phosphorylated in the absence of Ser108. (31)P NMR spectra of the S108A protein confirm that it is phosphorylated. Although the S108A/H329N protein had no detectable catalytic activity, the (31)P NMR spectra were not consistent with a phosphohistidine residue.     

Alkaline proteinase inhibitor of Pseudomonas aeruginosa: a mutational and molecular dynamics study of the role of N-terminal residues in the inhibition of Pseudomonas alkaline proteinase

Alkaline proteinase inhibitor of Pseudomonas aeruginosa is a 11.5-kDa, high affinity inhibitor of the serralysin class of zinc-dependent proteinases secreted by several Gram-negative bacteria. X-ray crystallography of the proteinase-inhibitor complex reveals that five N-terminal inhibitor residues occupy the extended substrate binding site of the enzyme and that the catalytic zinc is chelated by the alpha-amino and carbonyl groups of the N-terminal residue of the inhibitor. In this study, we assessed the effect of alteration of inhibitor residues 2-5 on its affinity for Pseudomonas alkaline proteinase (APR) as derived from the ratio of the dissociation and associate rate constants for formation of the enzyme-inhibitor complex. The largest effect was observed at position Ser-2, which occupies the S1' pocket of the enzyme and donates a hydrogen bond to the carboxyl group of the catalytic Glu-177 of the proteinase. Substitution of Asp, Arg, or Trp at this position increased the dissociation constant KD by 35-, 180-, and 13-fold, respectively. Mutation at positions 3-5 of the trunk also resulted in a reduction in enzyme-inhibitor affinity, with the exception of an I4W mutant, which exhibited a 3-fold increase in affinity. Molecular dynamics simulation of the complex formation between the catalytic domain of APR and the S2D mutant showed that the carboxyl of Asp-2 interacts with the catalytic zinc, thereby partially neutralizing the negative charge that otherwise would clash with the carboxyl group of Glu-177 of APR. Simulation of the interaction between the alkaline proteinase and the I4W mutant revealed a major shift in the loop comprised of residues 189-200 of the enzyme that allowed formation of a stacking interaction between the aromatic rings of Ile-4 of the inhibitor and Tyr-158 of the proteinase. This new interaction could account for the observed increase in enzyme-inhibitor affinity.     

Crystal structures of active fully assembled substrate- and product-bound complexes of UDP-N-acetylmuramic acid:L-alanine ligase (MurC) from Haemophilus influenzae

UDP-N-acetylmuramic acid:L-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg(2+) and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-L-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn(2+) have been determined to 1.85- and 1.7-A resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the gamma-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates.     

Three-dimensional structure of rat-liver acyl-CoA oxidase in complex with a fatty acid: insights into substrate-recognition and reactivity toward molecular oxygen

The three-dimensional structure of rat-liver acyl-CoA oxidase-II (ACO-II) in a complex with a C12-fatty acid was solved by the molecular replacement method based on the uncomplexed ACO-II structure. The crystalline form of the complex was obtained by cocrystallization of ACO-II with dodecanoyl-CoA. The crystalline complex possessed, in the active-site crevice, only the fatty acid moiety that had been formed through hydrolysis of the thioester bond. The overall dimeric structure and the folding pattern of each subunit are essentially superimposable on those of uncomplexed ACO-II. The active site including the flavin ring of FAD, the crevice embracing the fatty acyl moiety, and adjacent amino acid side chains are superimposably conserved with the exception of Glu421, whose carboxylate group is tilted away to accommodate the fatty acid. One of the carboxyl oxygens of the bound fatty acid is hydrogen-bonded to the amide hydrogen of Glu421, the presumed catalytic base, and to the ribityl 2'-hydroxyl group of FAD. This hydrogen-bonding network correlates well with the substrate recognition/activation in acyl-CoA dehydrogenase. The binding mode of C12-fatty acid suggests that the active site does not close upon substrate binding, but remains spacious during the entire catalytic process, the oxygen accessibility in the oxidative half-reaction thereby being maintained.     

Structural insights into the second step of RNA-dependent cysteine biosynthesis in archaea: crystal structure of Sep-tRNA:Cys-tRNA synthase from Archaeoglobus fulgidus

In the ancient organisms, methanogenic archaea, lacking the canonical cysteinyl-tRNA synthetase, Cys-tRNA(Cys) is produced by an indirect pathway, in which O-phosphoseryl-tRNA synthetase ligates O-phosphoserine (Sep) to tRNA(Cys) and Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNA(Cys) to Cys-tRNA(Cys). In this study, the crystal structure of SepCysS from Archaeoglobus fulgidus has been determined at 2.4 A resolution. SepCysS forms a dimer, composed of monomers bearing large and small domains. The large domain harbors the seven-stranded beta-sheet, which is typical of the pyridoxal 5'-phosphate (PLP)-dependent enzymes. In the active site, which is located near the dimer interface, PLP is covalently bound to the side-chain of the conserved Lys209. In the proximity of PLP, a sulfate ion is bound by the side-chains of the conserved Arg79, His103, and Tyr104 residues. The active site is located deep within the large, basic cleft to accommodate Sep-tRNA(Cys). On the basis of the surface electrostatic potential, the amino acid residue conservation mapping, the position of the bound sulfate ion, and the substrate amino acid binding manner in other PLP-dependent enzymes, a binding model of Sep-tRNA(Cys) to SepCysS was constructed. One of the three strictly conserved Cys residues (Cys39, Cys42, or Cys247), of one subunit may play a crucial role in the catalysis in the active site of the other subunit.     

Amphipathic benzoic acid derivatives: synthesis and binding in the hydrophobic tunnel of the zinc deacetylase LpxC

The first committed step in lipid A biosynthesis is catalyzed by uridine diphosphate-(3-O-(R-3-hydroxymyristoyl))-N-acetylglucosamine deacetylase (LpxC), a zinc-dependent deacetylase, and inhibitors of LpxC may be useful in the development of antibacterial agents targeting a broad spectrum of Gram-negative bacteria. Here, we report the design of amphipathic benzoic acid derivatives that bind in the hydrophobic tunnel in the active site of LpxC. The hydrophobic tunnel accounts for the specificity of LpxC toward substrates and substrate analogues bearing a 3-O-myristoyl substituent. Simple benzoic acid derivatives bearing an aliphatic 'tail' bind in the hydrophobic tunnel with micromolar affinity despite the lack of a glucosamine ring like that of the substrate. However, although these benzoic acid derivatives each contain a negatively charged carboxylate 'warhead' intended to coordinate to the active site zinc ion, the 2.25A resolution X-ray crystal structure of LpxC complexed with 3-(heptyloxy)benzoate reveals 'backward' binding in the hydrophobic tunnel, such that the benzoate moiety does not coordinate to zinc. Instead, it binds at the outer end of the hydrophobic tunnel. Interestingly, these ligands bind with affinities comparable to those measured for more complicated substrate analogue inhibitors containing glucosamine ring analogues and hydroxamate 'warheads' that coordinate to the active site zinc ion. We conclude that the intermolecular interactions in the hydrophobic tunnel dominate enzyme affinity in this series of benzoic acid derivatives.     

Amino acid discrimination by a class I aminoacyl-tRNA synthetase specified by negative determinants

The 2.5 A crystal structure of Escherichia coli glutaminyl-tRNA synthetase in a quaternary complex with tRNA(Gln), an ATP analog and glutamate reveals that the non-cognate amino acid adopts a distinct binding mode within the active site cleft. In contrast to the binding of cognate glutamine, one oxygen of the charged glutamate carboxylate group makes a direct ion-pair interaction with the strictly conserved Arg30 residue located in the first half of the dinucleotide fold domain. The nucleophilic alpha-carboxylate moiety of glutamate is mispositioned with respect to both the ATP alpha-phosphate and terminal tRNA ribose groups, suggesting that a component of amino acid discrimination resides at the catalytic step of the reaction. Further, the other side-chain carboxylate oxygen of glutamate is found in a position identical to that previously proposed to be occupied by the NH(2) group of the cognate glutamine substrate. At this position, the glutamate oxygen accepts hydrogen bonds from the hydroxyl moiety of Tyr211 and a water molecule. These findings demonstrate that amino acid specificity by GlnRS cannot arise from hydrogen bonds donated by the cognate glutamine amide to these same moieties, as previously suggested. Instead, Arg30 functions as a negative determinant to drive binding of non-cognate glutamate into a non-productive orientation. The poorly differentiated cognate amino acid-binding site in GlnRS may be a consequence of the late emergence of this enzyme from the eukaryotic lineage of glutamyl-tRNA synthetases.     

The donor substrate specificity of the human beta 1,3-glucuronosyltransferase I toward UDP-glucuronic acid is determined by two crucial histidine and arginine residues

The human beta1,3-glucuronosyltransferase I (GlcAT-I) plays a key role in proteoglycan biosynthesis by catalyzing the transfer of glucuronic acid onto the trisaccharide-protein linkage structure Galbeta1,3Galbeta1,4Xylbeta-O-Ser, a prerequisite step for polymerization of glycosaminoglycan chains. In this study, we identified His(308) and Arg(277) residues as essential determinants for the donor substrate (UDP-glucuronic acid) selectivity of the human GlcAT-I. Analysis of the UDP-glucuronic acid-binding site by computational modeling in conjunction with site-directed mutagenesis indicated that both residues interact with glucuronic acid. Substitution of His(308) by arginine induced major changes in the donor substrate specificity of GlcAT-I. Interestingly, the H308R mutant was able to efficiently utilize nucleotide sugars UDP-glucose, UDP-mannose, and UDP-N-acetylglucosamine, which are not naturally accepted by the wild-type enzyme, as co-substrate in the transfer reaction. To gain insight into the role of Arg(277), site-directed mutagenesis in combination with chemical modification was carried out. Substitution of Arg(277) with alanine abrogated the activity of GlcAT-I. Furthermore, the arginine-directed reagent 2,3-butanedione irreversibly inhibited GlcAT-I, which was effectively protected against inactivation by UDP-glucuronic acid but not by UDP-glucose. It is noteworthy that the activity of the H308R mutant toward UDP-glucose was unaffected by the arginine-directed reagent. Our results are consistent with crucial interactions between the His(308) and Arg(277) residues and the glucuronic acid moiety that governs the specificity of GlcAT-I toward the nucleotide sugar donor substrate.