Crystal structure of activated ribulose-1,5-bisphosphate carboxylase complexed with its substrate, ribulose-1,5-bisphosphate

The three-dimensional structure of the complex of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum, CO2, Mg2+, and ribulose bisphosphate has been determined with x-ray crystallographic methods to 2.6-A resolution. Ribulose-1,5-bisphosphate binds across the active site with the two phosphate groups in the two phosphate binding sites of the beta/alpha barrel. The oxygen atoms of the carbamate and the side chain of Asp-193 provide the protein ligands to the bound Mg2+ ion. The C2 and the C3 or C4 oxygen atoms of the substrate are also within the first coordination sphere of the metal ion. At the present resolution of the electron density maps, two slightly different conformations of the substrate, with the C3 hydroxyl group "cis" or "trans" to the C2 oxygen, can be built into the observed electron density. The two different conformations suggest two different mechanisms of proton abstraction in the first step of catalysis, the enolization of the ribulose 1,5-bisphosphate. Two loop regions, which are disordered in the crystals of the nonactivated enzyme, could be built into their respective electron density. A comparison with the structure of the quaternary complex of the spinach enzyme shows that despite the different conformations of loop 6, the positions of the Mg2+ ion, and most atoms of the substrate are very similar when superimposed on each other. There are, however, some significant differences at the active site, especially in the metal coordination sphere.     

NO binding to Mn-substituted homoprotocatechuate 2,3-dioxygenase: relationship to O2 reactivity

Iron(II)-containing homoprotocatechuate 2,3-dioxygenase (FeHPCD) activates O₂ to catalyze the aromatic ring opening of homoprotocatechuate (HPCA). The enzyme requires Fe^II for catalysis, but Mn^II can be substituted (MnHPCD) with essentially no change in the steady-state kinetic parameters. Near simultaneous O₂ and HPCA activation has been proposed to occur through transfer of an electron or electrons from HPCA to O₂ through the divalent metal. In O₂ reactions with MnHPCD–HPCA and the 4-nitrocatechol (4NC) complex of the His200Asn (H200N) variant of FeHPCD, this transfer has resulted in the detection of a transient M^III–O₂ ^·? species that is not observed during turnover of the wild-type FeHPCD. The factors governing formation of the M^III–O₂ ^·? species are explored here by EPR spectroscopy using MnHPCD and nitric oxide (NO) as an O₂ surrogate. Both the HPCA and the dihydroxymandelic substrate complexes of MnHPCD bind NO, thus representing the first reported stable MnNO complexes of a nonheme enzyme. In contrast, the free enzyme, the MnHPCD–4NC complex, and the MnH200N and MnH200Q variants with or without HPCA bound do not bind NO. The MnHPCD–ligand complexes that bind NO are also active in normal O₂-linked turnover, whereas the others are inactive. Past studies have shown that FeHPCD and the analogous variants and catecholic ligand complexes all bind NO, and are active in normal turnover. This contrasting behavior may stem from the ability of the enzyme to maintain the approximately 0.8-V difference in the solution redox potentials of Fe^II and Mn^II. Owing to the higher potential of Mn, the formation of the NO adduct or the O₂ adduct requires both strong charge donation from the bound catecholic ligand and additional stabilization by interaction with the active-site His200. The same nonoptimal electronic and structural forces that prevent NO and O₂ binding in MnHPCD variants may lead to inefficient electron transfer from the catecholic substrate to the metal center in variants of FeHPCD during O₂-linked turnover. Accordingly, past studies have shown that intermediate Fe^III species are observed for these mutant enzymes.     

The co-crystal structure of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) with a tripeptide fluoromethyl ketone (Z-VAE(OMe)-FMK)

UCHL1 is a 223 amino acid member of the UCH family of deubiquitinating enzymes (DUBs), found abundantly and exclusively expressed in neurons and the testis in normal tissues. Two naturally occurring variants of UCHL1 are directly involved in Parkinson’s disease (PD). Not only has UCHL1 been linked to PD, but it has oncogenic properties, having been found abnormally expressed in lung, pancreatic, and colorectal cancers. Although inhibitors of UCHL1 have been described previously the co-crystal structure of the enzyme bound to any inhibitor has not been reported. Herein, we report the X-ray structure of UCHL1 co-crystallized with a peptide-based fluoromethylketone inhibitor, Z-VAE(OMe)-FMK (VAEFMK) at 2.35 Å resolution. The co-crystal structure reveals that the inhibitor binds in the active-site cleft, irreversibly modifying the active-site cysteine; however, the catalytic histidine is still misaligned as seen in the native structure, suggesting that the inhibitor binds to an inactive form of the enzyme. Our structure also reveals that the inhibitor approaches the active-site cleft from the opposite side of the crossover loop as compared to the direction of approach of ubiquitin’s C-terminal tail, thereby occupying the P1′ (leaving group) site, a binding site perhaps used by the unknown C-terminal extension of ubiquitin in the actual in vivo substrate(s) of UCHL1. This structure provides a view of molecular contacts at the active-site cleft between the inhibitor and the enzyme as well as furnishing structural information needed to facilitate further design of inhibitors targeted to UCHL1 with high selectivity and potency.     

The conserved glycine/alanine residue of the active-site loop containing the putative acetylCoA-binding motif is essential for the overall structural integrity of Mesorhizobium loti arylamine N-acetyltransferase 1

The arylamine N-acetyltransferases are important xenobiotic-metabolizing enzymes that catalyze an acetyl group transfer from acetylCoA to arylamine substrates. NAT enzymes possess an active-site loop (the active-site P-loop) involved in substrate binding and selectivity. The Gly/Ala residue present at the start of the active-site P-loop, although conserved in all NAT enzymes, is not involved in the catalytic mechanism or substrate binding. Here we show that a small amino acid (such as Gly or Ala) at this position is important not only for maintaining the functions of the active-site P-loop but, more surprisingly, also important for maintaining the overall structural integrity of NAT enzymes. Our data thus suggest that in addition to its role in substrate binding and selectivity, the active-site P-loop could play a wider structural role in NAT enzymes.     

Escherichia coli methionine aminopeptidase: implications of crystallographic analyses of the native, mutant, and inhibited enzymes for the mechanism of catalysis.

By improving the expression and purification of Escherichia coli methionine aminopeptidase (eMetAP) and using slightly different crystallization conditions, the resolution of the parent structure was extended from 2.4 to 1.9 A resolution. This has permitted visualization of the coordination geometry and solvent structure of the active-site dinuclear metal center. One solvent molecule (likely a mu-hydroxide) bridges the trigonal bipyramidal (Co1) and octahedral (Co2) cobalt ions. A second solvent (possibly a hydroxide ion) is bound terminally to Co2. A monovalent cation binding site was also identified about 13 A away from the metal center at an interface between the two subdomains of the protein. The first structure of a substrate-like inhibitor, (3R)-amino-(2S)-hydroxyheptanoyl-L-Ala-L-Leu-L-Val-L-Phe-OMe, bound to a methionine aminopeptidase, has also been determined. This inhibitor coordinates the metal center through four interactions as follows: (i) ligation of the N-terminal (3R)-nitrogen to Co2, (ii, iii) bridging coordination of the (2S)-hydroxyl group, and (iv) terminal ligation to Co1 by the keto oxygen of the pseudo-peptide linkage. Inhibitor binding occurs with the displacement of two solvent ligands and the expansion of the coordination sphere of Co1. In addition to the tetradentate, bis-chelate metal coordination, the substrate analogue forms hydrogen bonds with His79 and His178, two conserved residues within the active site of all MetAPs. To evaluate their importance in catalysis His79 and His178 were replaced with alanine. Both substitutions, but especially that of His79, reduce activity. The structure of the His79Ala apoenzyme and the comparison of its electronic absorption spectra with other variants suggest that the loss in activity is not due to a conformational change or a defective metal center. Two different reaction mechanisms are proposed and are compared to those of related enzymes. These results also suggest that inhibitors analogous to that reported here may be useful in preventing angiogenesis in cancer and in the treatment of microbial and fungal infections.     

Magnetic resonance investigation of ionizable residues at the active site of thermolysin.

The details of the pH dependence of the thermodynamic and magnetic interactions of the active-site region of thermolysin in which manganese has replaced the active-site zinc atom and the inhibitor N-trifluoroacetyl-D-phenylalanine have been examined. These show a number of ionizable groups in the active-site region. A cooperative displacement of manganese at the catalytic site is observed as pH is lowered. This appears to be the result of the protonation of histidine-142 and -146 which act as metal ligands. The metal is 50% displaced at pH 6.0. At higher pH values, the environment of the bound manganese changes as a result of the ionization of at least two groups of approximate pKa = 8.5 and 9.5. These values are assigned to tyrosine-157 and to the water molecule which acts as a metal ligand at the active site. The binding behavior of the inhibitor strongly suggests that two molecules of inhibitor bind to the enzyme. The weaker site is competitive with the synthetic substrate FAGLA (furylacryloylglycyl-leucinamide), while the strong site has no effect on FAGLA hydrolysis. This second site is in the vicinity of the active site with a distance of 8 A or less between the trifluoromethyl group and manganese bound at the active site.     

Structure of peptidase T from Salmonella typhimurium.

The structure of peptidase T, or tripeptidase, was determined by multiple wavelength anomalous dispersion (MAD) methodology and refined to 2.4 A resolution. Peptidase T comprises two domains; a catalytic domain with an active site containing two metal ions, and a smaller domain formed through a long insertion into the catalytic domain. The two metal ions, presumably zinc, are separated by 3.3 A, and are coordinated by five carboxylate and histidine ligands. The molecular surface of the active site is negatively charged. Peptidase T has the same basic fold as carboxypeptidase G2. When the structures of the two enzymes are superimposed, a number of homologous residues, not evident from the sequence alone, could be identified. Comparison of the active sites of peptidase T, carboxypeptidase G2, Aeromonas proteolytica aminopeptidase, carboxypeptidase A and leucine aminopeptidase reveals a common structural framework with interesting similarities and differences in the active sites and in the zinc coordination. A putative binding site for the C-terminal end of the tripeptide substrate was found at a peptidase T specific fingerprint sequence motif.     

Crystal structure of D-psicose 3-epimerase from Agrobacterium tumefaciens and its complex with true substrate D-fructose: a pivotal role of metal in catalysis, an active site for the non-phosphorylated substrate, and its conformational changes

D-psicose, a rare sugar produced by the enzymatic reaction of D-tagatose 3-epimerase (DTEase), has been used extensively for the bioproduction of various rare carbohydrates. Recently characterized D-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was found to belong to the DTEase family and to catalyze the interconversion of D-fructose and D-psicose by epimerizing the C-3 position, with marked efficiency for D-psicose. The crystal structures of DPEase and its complex with the true substrate D-fructose were determined; DPEase is a tetramer and each monomer belongs to a TIM-barrel fold. The active site in each subunit is distinct from that of other TIM-barrel enzymes, which use phosphorylated ligands as the substrate. It contains a metal ion with octahedral coordination to two water molecules and four residues that are absolutely conserved across the DTEase family. Upon binding of D-fructose, the substrate displaces water molecules in the active site, with a conformation mimicking the intermediate cis-enediolate. Subsequently, Trp112 and Pro113 in the beta4-alpha4 loop undergo significant structural changes, sealing off the active site. Structural evidence and site-directed mutagenesis of the putative catalytic residues suggest that the metal ion plays a pivotal role in catalysis by anchoring the bound D-fructose, and Glu150 and Glu244 carry out an epimerization reaction at the C-3 position.     

An EXAFS study of the interaction of substrate with the ferric active site of protocatechuate 3,4-dioxygenase

X-ray crystallographic studies of the intradiol cleaving protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa have shown that the enzyme has a trigonal bipyramidal ferric active site with two histidines, two tyrosines, and a solvent molecule as ligands [Ohlendorf, D.H., Lipscomb, J.D., & Weber, P.C. (1988) Nature 336, 403-405]. Fe K-edge EXAFS studies of the spectroscopically similar protocatechuate 3,4-dioxygenase from Brevibacterium fuscum are consistent with a pentacoordinate geometry of the iron active site with 3 O/N ligands at 1.90 A and 2 O/N ligands at 2.08 A. The 2.08-A bonds are assigned to the two histidines, while the 1.90-A bonds are associated with the two tyrosines and the coordinated solvent. The short Fe-O distance for the solvent suggests that it coordinates as hydroxide rather than water. When the inhibitor terephthalate is bound to the enzyme, the XANES data indicate that the ferric site becomes 6-coordinate and the EXAFS data show a beat pattern which can only be simulated with an additional Fe-O/N interaction at 2.46 A. Together, the data suggest that the oxygens of the carboxylate group in terephthalate displace the hydroxide and chelate to the ferric site but in an asymmetric fashion. In contrast, protocatechuate 3,4-dioxygenase remains 5-coordinate upon the addition of the slow substrate homoprotocatechuic acid (HPCA). Previous EPR data have indicated that HPCA forms an iron chelate via the two hydroxyl functions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxy intermediates of homoprotocatechuate 2,3-dioxygenase: facile electron transfer between substrates

Substrates homoprotocatechuate (HPCA) and O(2) bind to the Fe(II) of homoprotocatechuate 2,3-dioxygenase (FeHPCD) in adjacent coordination sites. Transfer of an electron(s) from HPCA to O(2) via the iron is proposed to activate the substrates for reaction with each other to initiate aromatic ring cleavage. Here, rapid-freeze-quench methods are used to trap and spectroscopically characterize intermediates in the reactions of the HPCA complexes of FeHPCD and the variant His200Asn (FeHPCD?HPCA and H200N?HPCA, respectively) with O(2). A blue intermediate forms within 20 ms of mixing of O(2) with H200N?HPCA (H200N(Int1)(HPCA)). Parallel mode electron paramagnetic resonance and Mo?ssbauer spectroscopies show that this intermediate contains high-spin Fe(III) (S = 5/2) antiferromagnetically coupled to a radical (S(R) = 1/2) to yield an S = 2 state. Together, optical and Mo?ssbauer spectra of the intermediate support assignment of the radical as an HPCA semiquinone, implying that oxygen is bound as a (hydro)peroxo ligand. H200N(Int1)(HPCA) decays over the next 2 s, possibly through an Fe(II) intermediate (H200N(Int2)(HPCA)), to yield the product and the resting Fe(II) enzyme. Reaction of FeHPCD?HPCA with O(2) results in rapid formation of a colorless Fe(II) intermediate (FeHPCD(Int1)(HPCA)). This species decays within 1 s to yield the product and the resting enzyme. The absence of a chromophore from a semiquinone or evidence of a spin-coupled species in FeHPCD(Int1)(HPCA) suggests it is an intermediate occurring after O(2) activation and attack. The similar Mo?ssbauer parameters for FeHPCD(Int1)(HPCA) and H200N(Int2)(HPCA) suggest these are similar intermediates. The results show that transfer of an electron from the substrate to the O(2) via the iron does occur, leading to aromatic ring cleavage.     

The crystal structure of Escherichia coli class II fructose-1, 6-bisphosphate aldolase in complex with phosphoglycolohydroxamate reveals details of mechanism and specificity

The structure of a class II fructose-1,6-bisphosphate aldolase in complex with the substrate analogue and inhibitor phosphoglycolohydroxamate (PGH) has been determined using X-ray diffraction terms to a resolution of 2.0 A (1 A=0.1 nm). The crystals are trigonal, space group P3121 with a=b=78.24 A, c=289.69 A. The asymmetric unit is a homodimer of (alpha/beta)8 barrels and the model has refined to give R-work 19.2 %, R-free (based on 5 % of the data) 23.0 %. PGH resembles the ene-diolate transition state of the physiological substrate dihydroxyacetone phosphate. It is well ordered and bound in a deep polar cavity at the C-terminal end of the (alpha/beta)8 barrel, where it chelates the catalytic zinc ion using hydroxyl and enolate oxygen atoms. Trigonal bipyramidal coordination of the zinc ion is completed by three histidine residues. The complex network of hydrogen bonds at the catalytic centre is required to organise the position of key functional groups and metal ion ligands. A well-defined monovalent cation-binding site is observed following significant re-organisation of loop structures. This assists the formation of a phosphate-binding site on one side of the barrel that tethers PGH in the catalytic site. The positions of functional groups of substrate and putative interactions with key amino acid residues are identified. Knowledge of the complex structure complements the results of spectroscopic and site-directed mutagenesis studies, and contributes to our understanding of the mechanism and substrate specificity of this family of enzymes. A reaction mechanism distinct from that proposed for other class II aldolases is discussed. The results suggest that the class II aldolases should be sub-divided into two groups on the basis of both distinct folds and mechanism.     

Evidence that elongation of the catalytic loop of the Azotobacter vinelandii rhodanese changed selectivity from sulfur- to phosphate-containing substrates

Recent investigations have shown that the rhodanese domains, ubiquitous structural modules which might represent an example of conserved structures with possible functional diversity, are structurally related to the catalytic subunit of Cdc25 phosphatase enzymes. The major difference characterizing the active-site of the Azotobacter vinelandii rhodanese RhdA, with respect to the closely related Cdc25s (A, B, C), is that in Cdc25 phosphatases the active site loop [His-Cys-(X)5-Arg] is one residue longer than in RhdA [His-Cys-(X)4-Arg]. According to the hypothesis that the length of the RhdA active-site loop should play a key role in substrate recognition and catalytic activity, RhdA scaffold was the starting point for producing mutants with single-residue insertion to generate the catalytic loop HCQTHAHR (in RhdA-Ala) and HCQTHSHR (in RhdA-Ser). Analyses of the catalytic performances of the engineered RhdAs revealed that elongation of the catalytic loop definitely compromised the ability to catalyze sulfur transfer reactions, while it generated 'phosphatase' enzymes able to interact productively with the artificial substrate 3-O-methylfluorescein phosphate. Although this study is restricted to an example of rhodanese modules (RhdA), it provided experimental evidence of the hypothesis that a specific mutational event (a single-residue insertion or deletion in the active-site loop) could change the selectivity from sulfur- to phosphate-containing substrates (or vice versa).     

Crystal structure of ATP sulfurylase from the bacterial symbiont of the hydrothermal vent tubeworm Riftia pachyptila.

In sulfur chemolithotrophic bacteria, the enzyme ATP sulfurylase functions to produce ATP and inorganic sulfate from APS and inorganic pyrophosphate, which is the final step in the biological oxidation of hydrogen sulfide to sulfate. The giant tubeworm, Riftia pachyptila, which lives near hydrothermal vents on the ocean floor, harbors a sulfur chemolithotroph as an endosymbiont in its trophosome tissue. This yet-to-be-named bacterium was found to contain high levels of ATP sulfurylase that may provide a substantial fraction of the organisms ATP. We present here, the crystal structure of ATP sulfurylase from this bacterium at 1.7 A resolution. As predicted from sequence homology, the enzyme folds into distinct N-terminal and catalytic domains, but lacks the APS kinase-like C-terminal domain that is present in fungal ATP sulfurylase. The enzyme crystallizes as a dimer with one subunit in the crystallographic asymmetric unit. Many buried solvent molecules mediate subunit contacts at the interface. Despite the high concentration of sulfate needed for crystallization, no ordered sulfate was observed in the sulfate-binding pocket. The structure reveals a mobile loop positioned over the active site. This loop is in a "closed" or "down" position in the reported crystal structures of fungal ATP sulfurylases, which contained bound substrates, but it is in an "open" or "up" position in the ligand-free Riftia symbiont enzyme. Thus, closure of the loop correlates with occupancy of the active site, although the loop itself does not interact directly with bound ligands. Rather, it appears to assist in the orientation of residues that do interact with active-site ligands. Amino acid differences between the mobile loops of the enzymes from sulfate assimilators and sulfur chemolithotrophs may account for the significant kinetic differences between the two classes of ATP sulfurylase.     

Aromatic ring cleavage by homoprotocatechuate 2,3-dioxygenase: role of His200 in the kinetics of interconversion of reaction cycle intermediates

Homoprotocatechuate 2,3-dioxygenase (WT 2,3-HPCD) isolated from Brevibacterium fuscum utilizes an active site Fe(II) and O(2) to catalyze proximal extradiol cleavage of the aromatic ring of the substrate (HPCA). Here, the conserved active site residue His200 is changed to Gln, Glu, Ala, Asn, and Phe, and the reactions of the mutant enzymes are probed using steady-state and transient kinetic techniques. Each mutant catalyzes ring cleavage of HPCA to yield the normal product. H200Q and H200N retain 30-40% of the WT 2,3-HPCD activity at 24 degrees C, but the other mutants reduce the k(cat) to less than 9% of normal. The origin of the reduced activity is unlikely to be the substrate binding phase of the catalytic cycle, because the multistep anaerobic binding reaction of the chromophoric substrate 4-nitrocatechol (4NC) is shown to proceed with rate constants similar to those observed for WT 2,3-HPCD. In contrast, the rate constants of several steps in the multistep O(2) binding/insertion and product release half of the reaction cycle are substantially slowed, in particular the steps in which activated oxygen attacks the organic substrate and in which product is released. In the case of the H200N mutant, the product of 4NC oxidation is not the usual ring cleavage product, but rather the 4NC quinone. These results suggest that the main role of His200 is in facilitating the steps in the second half of the reaction cycle. The decreased rate constants for the O(2) insertion steps in the catalytic cycles of the mutant enzymes allow the oxygen adduct of an extradiol dioxygenase to be detected for the first time.     

A two-electron-shell game: intermediates of the extradiol-cleaving catechol dioxygenases

Extradiol-cleaving catechol dioxygenases function by binding both the organic substrate and O₂ at a divalent metal center in the active site. They have proven to be a particularly versatile group of enzymes with which to study the O₂ activation process. Here, recent studies of homoprotocatechuate 2,3-dioxygenase are summarized, showing how nature can utilize the enzyme structure and the properties of the metal and the substrate to select among many possible chemical paths to achieve both specificity and efficiency. Possible intermediates in the mechanism have been trapped by swapping active-site metals, introducing active-site amino acid substituted variants, and using substrates with different electron-donating capacities. Although each of these intermediates could form part of a viable reaction pathway, kinetic measurements significantly limit the likely candidates. Structural, kinetic, spectroscopic, and computational analyses of the various intermediates shed light on how catalytic efficiency can be achieved.     

Crystal structure of Escherichia coli PdxA,an enzyme involved in the pyridoxal phosphate biosynthesis pathway

Pyridoxal 5'-phosphate is an essential cofactor for many enzymes responsible for the metabolic conversions of amino acids. Two pathways for its de novo synthesis are known. The pathway utilized by Escherichia coli consists of six enzymatic steps catalyzed by six different enzymes. The fourth step is catalyzed by 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA, E.C. 1.1.1.262), which converts 4-hydroxy-l-threonine phosphate (HTP) to 3-amino-2-oxopropyl phosphate. This divalent metal ion-dependent enzyme has a strict requirement for the phosphate ester form of the substrate HTP, but can utilize either NADP+ or NAD+ as redox cofactor. We report the crystal structure of E. coli PdxA and its complex with HTP and Zn2+. The protein forms tightly bound dimers. Each monomer has an alpha/beta/alpha-fold and can be divided into two subdomains. The active site is located at the dimer interface, within a cleft between the two subdomains and involves residues from both monomers. A Zn2+ ion is bound within each active site, coordinated by three conserved histidine residues from both monomers. In addition two conserved amino acids, Asp247 and Asp267, play a role in maintaining integrity of the active site. The substrate is anchored to the enzyme by the interactions of its phospho group and by coordination of the amino and hydroxyl groups by the Zn2+ ion. PdxA is structurally similar to, but limited in sequence similarity with isocitrate dehydrogenase and isopropylmalate dehydrogenase. These structural similarities and the comparison with a NADP-bound isocitrate dehydrogenase suggest that the cofactor binding mode of PdxA is very similar to that of the other two enzymes and that PdxA catalyzes a stepwise oxidative decarboxylation of the substrate HTP.     

Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination.

A combination of half-site substrates and step arrest mutants of Flp, a site-specific recombinase of the integrase family, had earlier revealed the following features of the half-site recombination reaction. (i) The Flp active site is assembled by sharing of catalytic residues from at least two monomers of the protein. (ii) A Flp monomer does not cleave the half site to which it is bound (DNA cleavage in cis); rather, it cleaves a half site bound by a second Flp monomer (DNA cleavage in trans). For the lambda integrase (Int protein), the prototype member of the Int family, catalytic complementation between two active-site mutants has been observed in reactions with a suicide attL substrate. By analogy with Flp, this observation is strongly suggestive of a shared active site and of trans DNA cleavage. However, reactions with linear suicide attB substrates and synthetic Holliday junctions are more compatible with cis than with trans DNA cleavage. These Int results either argue against a common mode of active-site assembly within the Int family or challenge the validity of Flp half sites as mimics of the normal full-site substrates. We devised a strategy to assay catalytic complementation between Flp monomers in full sites. We found that the full-site reaction follows the shared active-site paradigm and the trans mode of DNA cleavage. These results suggest that within the Int family, a unitary chemical mechanism of recombination is achieved by more than one mode of physical interaction among the recombinase monomers.     

Structural Insights into the Substrate Specificity of (S)-Ureidoglycolate Amidohydrolase and Its Comparison with Allantoate Amidohydrolase

In plants, the ureide pathway is a metabolic route that converts the ring nitrogen atoms of purine into ammonia via sequential enzymatic reactions, playing an important role in nitrogen recovery. In the final step of the pathway, (S)-ureidoglycolate amidohydrolase (UAH) catalyzes the conversion of (S)-ureidoglycolate into glyoxylate and releases two molecules of ammonia as by-products. UAH is homologous in structure and sequence with allantoate amidohydrolase (AAH), an upstream enzyme in the pathway with a similar function as that of an amidase but with a different substrate. Both enzymes exhibit strict substrate specificity and catalyze reactions in a concerted manner, resulting in purine degradation. Here, we report three crystal structures of Arabidopsis thaliana UAH (bound with substrate, reaction intermediate, and product) and a structure of Escherichia coli AAH complexed with allantoate. Structural analyses of UAH revealed a distinct binding mode for each ligand in a bimetal reaction center with the active site in a closed conformation. The ligand directly participates in the coordination shell of two metal ions and is stabilized by the surrounding residues. In contrast, AAH, which exhibits a substrate-binding site similar to that of UAH, requires a larger active site due to the additional ureido group in allantoate. Structural analyses and mutagenesis revealed that both enzymes undergo an open-to-closed conformational transition in response to ligand binding and that the active-site size and the interaction environment in UAH and AAH are determinants of the substrate specificities of these two structurally homologous enzymes.     

Structure of the prolidase from Pyrococcus furiosus

The structure of prolidase from the hyperthermophilic archaeon Pyrococcus furiosus (Pfprol) has been solved and refined at 2.0 A resolution. This is the first structure of a prolidase, i.e., a peptidase specific for dipeptides having proline as the second residue. The asymmetric unit of the crystals contains a homodimer of the enzyme. Each of the two protein subunits has two domains. The C-terminal domain includes the catalytic site, which is centered on a dinuclear metal cluster. In the as-isolated form of Pfprol, the active-site metal atoms are Co(II) [Ghosh, M., et al. (1998) J. Bacteriol. 180, 4781-9]. An unexpected finding is that in the crystalline enzyme the active-site metal atoms are Zn(II), presumably as a result of metal exchange during crystallization. Both of the Zn(II) atoms are five-coordinate. The ligands include a bridging water molecule or hydroxide ion, which is likely to act as a nucleophile in the catalytic reaction. The two-domain polypeptide fold of Pfprol is similar to the folds of two functionally related enzymes, aminopeptidase P (APPro) and creatinase. In addition, the catalytic C-terminal domain of Pfprol has a polypeptide fold resembling that of the sole domain of a fourth enzyme, methionine aminopeptidase (MetAP). The active sites of APPro and MetAP, like that of Pfprol, include a dinuclear metal center. The metal ligands in the three enzymes are homologous. Comparisons with the molecular structures of APPro and MetAP suggest how Pfprol discriminates against oligopeptides and in favor of Xaa-Pro substrates. The crystal structure of Pfprol was solved by multiple-wavelength anomalous dispersion. The crystals yielded diffraction data of relatively high quality and resolution, despite the fact that one of the two protein subunits in the asymmetric unit was found to be significantly disordered. The final R and R(free) values are 0.24 and 0.28, respectively.