Structures of ligand-free and inhibitor complexes of dihydroorotase from Escherichia coli: implications for loop movement in inhibitor design

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamyl-L-aspartate (CA-asp) to L-dihydroorotate (DHO) in the de novo biosynthesis of pyrimidine nucleotides. DHOase is a potential anti-malarial drug target as malarial parasites can only synthesize pyrimidines via the de novo pathway and do not possess a salvage pathway. Here we report the structures of Escherichia coli DHOase crystallized without ligand (1.7 A resolution) and in the presence of the inhibitors 2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylate (HDDP; 2.0 A) and 5-fluoroorotate (FOA, 2.2 A). These are the first crystal structures of DHOase-inhibitor complexes, providing structural information on the mode of inhibitor binding. HDDP possesses features of both the substrate and product, and ligates the Zn atoms in the active site. In addition, HDDP forms hydrogen bonds to the flexible loop (residues 105-115) stabilizing the "loop-in" conformation of the flexible loop normally associated with the presence of CA-asp in the active site. By contrast, FOA, a product-like inhibitor, binds to the active site in a similar fashion to DHO but does not ligate the Zn atoms directly nor stabilize the loop-in conformation. These structures define the necessary features for the future design of improved inhibitors of DHOase.     

Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase)

Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine synthesis pathway and is responsible for the reversible cyclization of carbamyl-aspartate (Ca-asp) to dihydroorotate (DHO). DHOase is further divided into two classes based on several structural characteristics, one of which is the length of the flexible catalytic loop that interacts with the substrate, Ca-asp, regulating the enzyme activity. Here, we present the crystal structure of Class I Bacillus anthracis DHOase with Ca-asp in the active site, which shows the peptide backbone of glycine in the shorter loop forming the necessary hydrogen bonds with the substrate, in place of the two threonines found in Class II DHOases. Despite the differences in the catalytic loop, the structure confirms that the key interactions between the substrate and active site residues are similar between Class I and Class II DHOase enzymes, which we further validated by mutagenesis studies. B. anthracis DHOase is also a potential antibacterial drug target. In order to identify prospective inhibitors, we performed high-throughput screening against several libraries using a colorimetric enzymatic assay and an orthogonal fluorescence thermal binding assay. Surface plasmon resonance was used for determining binding affinity (K_D) and competition analysis with Ca-asp. Our results highlight that the primary difference between Class I and Class II DHOase is the catalytic loop. We also identify several compounds that can potentially be further optimized as potential B. anthracis inhibitors.     

13C and 15N isotope effects for conversion of L-dihydroorotate to N-carbamyl-L-aspartate using dihydroorotase from hamster and Bacillus caldolyticus

In the pyrimidine biosynthetic pathway, N-carbamyl-L-aspartate (CA-asp) is converted to L-dihydroorotate (DHO) by dihydroorotase (DHOase). The mechanism of this important reaction was probed using primary and secondary 15N and 13C isotope effects on the ring opening of DHO using isotope ratio mass spectrometry (IRMS). The reaction was performed at three different temperatures (25, 37, and 45 degrees C for hamster DHOase; 37, 50, and 60 degrees C for Bacillus caldolyticus), and the product CA-asp was purified for analysis. The primary and secondary kinetic isotope effects for the ring opening of the DHO were determined from analysis of the N and C of the carbamyl group after hydrolysis. In addition, the beta-carboxyl of the residual aspartate was liberated enzymatically by transamination to oxaloacetate with aspartate aminotransferase and then decarboxylation with oxaloacetate decarboxylase. The 13C/12C ratio from the released CO2 was determined by IRMS, yielding a second primary isotope effect. The primary and secondary isotope effects for the reaction catalyzed by DHOase showed little variation between enzymes or temperatures, the primary 13C and 15N isotope effects being approximately 1% on average, while the secondary 13C isotope effect is negligible or very slightly normal (>1.0000). These data indicate that the chemistry is at least partially rate-limiting while the secondary isotope effects suggest that the transition state may have lost some bending and torsional modes leading to a slight lessening of bond stiffness at the carbonyl carbon of the amide of CA-asp. The equilibrium isotope effects for DHO --> CA-asp have also been measured (secondary 13K(eq) = 1.0028 +/- 0.0002, primary 13K(eq) = 1.0053 +/- 0.0003, primary 15K(eq) = 1.0027 +/- 0.0003). Using these equilibrium isotope effects, the kinetic isotope effects for the physiological reaction (CA-asp --> DHO) have been calculated. These values indicate that the carbon of the amide group is more stiffly bonded in DHO while the slightly lesser, but still normal, values of the primary kinetic isotope effect show that the chemistry remains at least partially rate-limiting for the physiological reaction. It appears that the ring opening and closing is the slow step of the reaction.     

Mechanism of the dihydroorotase reaction

Dihydroorotase (DHO) is a zinc metalloenzyme that functions in the pathway for the biosynthesis of pyrimidine nucleotides by catalyzing the reversible interconversion of carbamoyl aspartate and dihydroorotate. A chemical mechanism was proposed on the basis of an analysis of the effects of pH, metal substitution, solvent isotope effects, mutant proteins, and alternative substrates on the enzyme-catalyzed reaction. The pH-rate profiles for the hydrolysis of dihydroorotate or thiodihydroorotate demonstrated that a single group from the enzyme must be unprotonated for maximal catalytic activity. Conversely, the pH-rate profiles for the condensation of carbamoyl aspartate to dihydroorotate showed that a single group from the enzyme must be protonated for maximal catalytic activity. The native zinc ions within the active site of DHO were substituted with cobalt or cadmium by reconstitution of the apoenzyme with divalent cations in the presence of bicarbonate. The ionizations observed in the pH-rate profiles were dependent on the specific metal ion bound to the active site. Mutation of the residue (Asp-250) that hydrogen bonds to the bridging hydroxide (or water) resulted in the loss of catalytic activity. These results are consistent with the formation of a hydroxide bridge between the two divalent cations that functions as the nucleophile during the hydrolysis of dihydroorotate. In addition, Asp-250 is postulated to shuttle the proton from the bridging hydroxide to the leaving group amide during hydrolysis of dihydroorotate. The X-ray crystal structure of DHO showed that the exocyclic alpha-carboxylate of dihydroorotate is bound to the protein via electrostatic interactions with Arg-20, Asn-44, and His-254. Mutation of these residues resulted in the loss of catalytic activity, indicating that these residues are critical for substrate recognition. The thio analogue of dihydroorotate was found to be a good substrate of the enzyme. A comprehensive chemical mechanism for DHO was proposed on the basis of the experimental findings in this study and the X-ray crystal structure.     

Structural basis for the catalytic mechanism of aspartate ammonia lyase

Aspartate ammonia lyases (or aspartases) catalyze the reversible deamination of L-aspartate into fumarate and ammonia. The lack of crystal structures of complexes with substrate, product, or substrate analogues so far precluded determination of their precise mechanism of catalysis. Here, we report crystal structures of AspB, the aspartase from Bacillus sp. YM55-1, in an unliganded state and in complex with L-aspartate at 2.4 and 2.6 ? resolution, respectively. AspB forces the bound substrate to adopt a high-energy, enediolate-like conformation that is stabilized, in part, by an extensive network of hydrogen bonds between residues Thr101, Ser140, Thr141, and Ser319 and the substrate's β-carboxylate group. Furthermore, substrate binding induces a large conformational change in the SS loop (residues G(317)SSIMPGKVN(326)) from an open conformation to one that closes over the active site. In the closed conformation, the strictly conserved SS loop residue Ser318 is at a suitable position to act as a catalytic base, abstracting the Cβ proton of the substrate in the first step of the reaction mechanism. The catalytic importance of Ser318 was confirmed by site-directed mutagenesis. Site-directed mutagenesis of SS loop residues, combined with structural and kinetic analysis of a stable proteolytic AspB fragment, further suggests an important role for the small C-terminal domain of AspB in controlling the conformation of the SS loop and, hence, in regulating catalytic activity. Our results provide evidence supporting the notion that members of the aspartase/fumarase superfamily use a common catalytic mechanism involving general base-catalyzed formation of a stabilized enediolate intermediate.     

Structure of Salmonella typhimurium OMP Synthase in a Complete Substrate Complex

Dimeric Salmonella typhimurium orotate phosphoribosyltransferase (OMP synthase, EC 2.4.2.10), a key enzyme in de novo pyrimidine nucleotide synthesis, has been cocrystallized in a complete substrate E·MgPRPP·orotate complex and the structure determined to 2.2 ? resolution. This structure resembles that of Saccharomyces cerevisiae OMP synthase in showing a dramatic and asymmetric reorganization around the active site-bound ligands but shares the same basic topology previously observed in complexes of OMP synthase from S. typhimurium and Escherichia coli. The catalytic loop (residues 99-109) contributed by subunit A is reorganized to close the active site situated in subunit B and to sequester it from solvent. Furthermore, the overall structure of subunit B is more compact, because of movements of the amino-terminal hood and elements of the core domain. The catalytic loop of subunit B remains open and disordered, and subunit A retains the more relaxed conformation observed in loop-open S. typhimurium OMP synthase structures. A non-proline cis-peptide formed between Ala71 and Tyr72 is seen in both subunits. The loop-closed catalytic site of subunit B reveals that both the loop and the hood interact directly with the bound pyrophosphate group of PRPP. In contrast to dimagnesium hypoxanthine-guanine phosphoribosyltransferases, OMP synthase contains a single catalytic Mg(2+) in the closed active site. The remaining pyrophosphate charges of PRPP are neutralized by interactions with Arg99A, Lys100B, Lys103A, and His105A. The new structure confirms the importance of loop movement in catalysis by OMP synthase and identifies several additional movements that must be accomplished in each catalytic cycle. A catalytic mechanism based on enzymic and substrate-assisted stabilization of the previously documented oxocarbenium transition state structure is proposed.     

Intersubunit communication in the dihydroorotase–aspartate transcarbamoylase complex of Aquifex aeolicus

Aspartate transcarbamoylase and dihydroorotase, enzymes that catalyze the second and third step in de novo pyrimidine biosynthesis, are associated in dodecameric complexes in Aquifex aeolicus and many other organisms. The architecture of the dodecamer is ideally suited to channel the intermediate, carbamoyl aspartate from its site of synthesis on the ATC subunit to the active site of DHO, which catalyzes the next step in the pathway, because both reactions occur within a large, internal solvent‐filled cavity. Channeling usually requires that the reactions of the enzymes are coordinated so that the rate of synthesis of the intermediate matches its rate of utilization. The linkage between the ATC and DHO subunits was demonstrated by showing that the binding of the bisubstrate analog, N‐phosphonacetyl‐L ‐aspartate to the ATC subunit inhibits the activity of the distal DHO subunit. Structural studies identified a DHO loop, loop A, interdigitating between the ATC domains that would be expected to interfere with domain closure essential for ATC catalysis. Mutation of the DHO residues in loop A that penetrate deeply between the two ATC domains inhibits the ATC activity by interfering with the normal reciprocal linkage between the two enzymes. Moreover, a synthetic peptide that mimics that part of the DHO loop that binds between the two ATC domains was found to be an allosteric or noncompletive ATC inhibitor (K_i = 22 μM). A model is proposed suggesting that loop A is an important component of the functional linkage between the enzymes.     

Characterization of Helicobacter pylori gamma-glutamyltranspeptidase reveals the molecular basis for substrate specificity and a critical role for the tyrosine 433-containing loop in catalysis

Helicobacter pylori gamma-glutamyltranspeptidase (HpGT) is a member of the N-terminal nucleophile hydrolase superfamily. It is translated as an inactive 60 kDa polypeptide precursor that undergoes intramolecular autocatalytic cleavage to generate a fully active heterodimer composed of a 40 kDa and a 20 kDa subunit. The resultant N-terminus, Thr 380, has been shown to be the catalytic nucleophile in both autoprocessing and enzymatic reactions. Once processed, HpGT catalyzes the hydrolysis of the gamma-glutamyl bond in glutathione and its conjugates. To facilitate the determination of physiologically relevant substrates for the enzyme, crystal structures of HpGT in complex with glutamate (1.6 A, Rfactor = 16.7%, Rfree = 19.0%) and an inactive HpGT mutant, T380A, in complex with S-(nitrobenzyl)glutathione (1.55 A, Rfactor = 18.7%, Rfree = 21.8%) have been determined. Residues that comprise the gamma-glutamyl binding site are primarily located in the 20 kDa subunit and make numerous hydrogen bonds with the alpha-amino and alpha-carboxylate groups of the substrate. In contrast, a single hydrogen bond occurs between the T380A mutant and the remainder of the ligand. Lack of specific coordination beyond the gamma-glutamyl moiety may account for the substrate binding permissiveness of the enzyme. Structural analysis was combined with site-directed mutagenesis of residues involved in maintaining the conformation of a loop region that covers the gamma-glutamyl binding site. Results provide evidence that access to this buried site may occur through conformational changes in the Tyr 433-containing loop, as disruption of the intricate hydrogen-bond network responsible for optimal placement of Tyr 433 significantly diminishes catalytic activity.     

Aquifex aeolicus dihydroorotase: association with aspartate transcarbamoylase switches on catalytic activity

Dihydroorotase (DHOase) catalyzes the reversible condensation of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis. The enzyme from Aquifex aeolicus, a hyperthermophilic organism of ancient lineage, was cloned and expressed in Escherichia coli. The purified protein was found to be a 45-kDa monomer containing a single zinc ion. Although there is no other DHOase gene in the A. aeolicus genome, the recombinant protein completely lacked catalytic activity at any temperature tested. However, DHOase formed an active complex with aspartate transcarbamoylase (ATCase) from the same organism. Whereas the k(cat) of 13.8 +/- 0.03 s(-1) was close to the value observed for the mammalian enzyme, the K (m)for dihydroorotate, 3.03 +/- 0.05 mM was 433-fold higher. Gel filtration and chemical cross-linking showed that the complex exists as a 240-kDa hexamer (DHO(3)-ATC(3)) and a 480-kDa duodecamer (DHO(6)-ATC(6)) probably in rapid equilibrium. Complex formation protects both DHOase and ATCase against thermal degradation at temperatures near 100 degrees C where the organism grows optimally. These results lead to the reclassification of both enzymes: ATCase, previously considered a Class C homotrimer, now falls into Class A, whereas the DHOase is a Class 1B enzyme. CD spectroscopy indicated that association with ATCase does not involve a significant perturbation of the DHOase secondary structure, but the visible absorption spectrum of a Co(2+)-substituted DHOase is appreciably altered upon complex formation suggesting a change in the electronic environment of the active site. The association of DHOase with ATCase probably serves as a molecular switch that ensures that free, uncomplexed DHOase in the cell remains inactive. At pH 7.4, the equilibrium ratio of carbamoyl aspartate to dihydroorotate is 17 and complex formation may drive the reaction in the biosynthetic direction.     

Crystal structure analysis,covalent docking,and molecular dynamics calculations reveal a conformational switch in PhaZ7 PHB depolymerase

An open and a closed conformation of a surface loop in PhaZ7 extracellular poly(3‐hydroxybutyrate) depolymerase were identified in two high‐resolution crystal structures of a PhaZ7 Y105E mutant. Molecular dynamics (MD) simulations revealed high root mean square fluctuations (RMSF) of the 281–295 loop, in particular at residue Asp289 (RMSF 7.62 Å). Covalent docking between a 3‐hydroxybutyric acid trimer and the catalytic residue Ser136 showed that the binding energy of the substrate is significantly more favorable in the open loop conformation compared to that in the closed loop conformation. MD simulations with the substrate covalently bound depicted 1 Å RMSF higher values for the residues 281–295 in comparison to the apo (substrate‐free) form. In addition, the presence of the substrate in the active site enhanced the ability of the loop to adopt a closed form. Taken together, the analysis suggests that the flexible loop 281–295 of PhaZ7 depolymerase can act as a lid domain to control substrate access to the active site of the enzyme. Proteins 2017; 85:1351–1361. © 2017 Wiley Periodicals, Inc.     

A double-headed cathepsin B inhibitor devoid of warhead

Most synthetic inhibitors of peptidases have been targeted to the active site for inhibiting catalysis through reversible competition with the substrate or by covalent modification of catalytic groups. Cathepsin B is unique among the cysteine peptidase for the presence of a flexible segment, known as the occluding loop, which can block the primed subsites of the substrate binding cleft. With the occluding loop in the open conformation cathepsin B acts as an endopeptidase, and it acts as an exopeptidase when the loop is closed. We have targeted the occluding loop of human cathepsin B at its surface, outside the catalytic center, using a high-throughput docking procedure. The aim was to identify inhibitors that would interact with the occluding loop thereby modulating enzyme activity without the help of chemical warheads against catalytic residues. From a large library of compounds, the in silico approach identified [2-[2-(2,4-dioxo-1,3-thiazolidin-3-yl)ethylamino]-2-oxoethyl] 2-(furan-2-carbonylamino) acetate, which fulfills the working hypothesis. This molecule possesses two distinct binding moieties and behaves as a reversible, double-headed competitive inhibitor of cathepsin B by excluding synthetic and protein substrates from the active center. The kinetic mechanism of inhibition suggests that the occluding loop is stabilized in its closed conformation, mainly by hydrogen bonds with the inhibitor, thus decreasing endoproteolytic activity of the enzyme. Furthermore, the dioxothiazolidine head of the compound sterically hinders binding of the C-terminal residue of substrates resulting in inhibition of the exopeptidase activity of cathepsin B in a physiopathologically relevant pH range.     

Structure of Plasmodium falciparum triose-phosphate isomerase-2-phosphoglycerate complex at 1.1-A resolution

Triose-phosphate isomerase, a key enzyme of the glycolytic pathway, catalyzes the isomerization of dihydroxy acetone phosphate and glyceraldehyde 3-phosphate. In this communication we report the crystal structure of Plasmodium falciparum triose-phosphate isomerase complexed to the inhibitor 2-phosphoglycerate at 1.1-A resolution. The crystallographic asymmetric unit contains a dimeric molecule. The inhibitor bound to one of the subunits in which the flexible catalytic loop 6 is in the open conformation has been cleaved into two fragments presumably due to radiation damage. The cleavage products have been tentatively identified as 2-oxoglycerate and meta-phosphate. The intact 2-phosphoglycerate bound to the active site of the other subunit has been observed in two different orientations. The active site loop in this subunit is in both open and "closed" conformations, although the open form is predominant. Concomitant with the loop closure, Phe-96, Leu-167, and residues 208-211 (YGGS) are also observed in dual conformations in the B-subunit. Detailed comparison of the active-site geometry in the present case to the Saccharomyces cerevisiae triose-phosphate isomerase-dihydroxy acetone phosphate and Leishmania mexicana triose-phosphate isomerase-phosphoglycolate complexes, which have also been determined at atomic resolution, shows that certain interactions are common to the three structures, although 2-phosphoglycerate is neither a substrate nor a transition state analogue.     

Protein engineering of chymosin; modification of the optimum pH of enzyme catalysis

The aspartic proteinase chymosin exhibits a local network of hydrogen bonds involving the active site aspartates and surrounding residues which may have an influence on the rate and optimal pH of substrate cleavage. We have introduced into chymosin B the following substitutions: Asp304 to Ala (D304A), Thr218 to Ala (T218A) and Gly244 to Asp (G244D, chymosin A), using oligonucleotide-directed mutagenesis. Kinetic analysis of these active mutants shows shifts in their pH optima to 4.4 D304A, 4.2 T218A and 4.0 G244D compared with 3.8 for chymosin B using a synthetic octapeptide substrate. The upward shift of the D304A and T218A may be due to the loss of hydrogen bond interactions indirectly affecting the catalytic aspartates 32 and 215. The G244D mutation which is in a flexible loop on the surface of the enzyme may alter the conformation of the specificity pockets on the prime side of the scissile bond.     

Structure of methylene-tetrahydromethanopterin dehydrogenase from methylobacterium extorquens AM1

NADP-dependent methylene-H(4)MPT dehydrogenase, MtdA, from Methylobacterium extorquens AM1 catalyzes the dehydrogenation of methylene-tetrahydromethanopterin and methylene-tetrahydrofolate with NADP(+) as cosubstrate. The X-ray structure of MtdA with and without NADP bound was established at 1.9 A resolution. The enzyme is present as a homotrimer. The alpha,beta fold of the monomer is related to that of methylene-H(4)F dehydrogenases, suggesting a common evolutionary origin. The position of the active site is located within a large crevice built up by the two domains of one subunit and one domain of a second subunit. Methylene-H(4)MPT could be modeled into the cleft, and crucial active site residues such as Phe18, Lys256, His260, and Thr102 were identified. The molecular basis of the different substrate specificities and different catalytic demands of MtdA compared to methylene-H(4)F dehydrogenases are discussed.     

Molecular modeling of the dimeric structure of human lipoprotein lipase and functional studies of the carboxyl-terminal domain.

Lipoprotein lipase (LPL) plays a key role in lipid metabolism. Molecular modeling of dimeric LPL was carried out using insight ii based upon the crystal structures of human, porcine, and horse pancreatic lipase. The dimeric model reveals a saddle-shaped structure and the key heparin-binding residues in the amino-terminal domain located on the top of this saddle. The models of two dimeric conformations - a closed, inactive form and an open, active form - differ with respect to how surface-loop positions affect substrate access to the catalytic site. In the closed form, the surface loop covers the catalytic site, which becomes inaccessible to solvent. Large conformational changes in the open form, especially in the loop and carboxyl-terminal domain, allow substrate access to the active site. To dissect the structure-function relationships of the LPL carboxyl-terminal domain, several residues predicted by the model structure to be essential for the functions of heparin binding and substrate recognition were mutagenized. Arg405 plays an important role in heparin binding in the active dimer. Lys413/Lys414 or Lys414 regulates heparin affinity in both monomeric and dimeric forms. To evaluate the prediction that LPL forms a homodimer in a 'head-to-tail' orientation, two inactive LPL mutants - a catalytic site mutant (S132T) and a substrate-recognition mutant (W390A/W393A/W394A) - were cotransfected into COS7 cells. Lipase activity could be recovered only when heterodimerization occurred in a head-to-tail orientation. After cotransfection, 50% of the wild-type lipase activity was recovered, indicating that lipase activity is determined by the interaction between the catalytic site on one subunit and the substrate-recognition site on the other.     

Crystal structure of phospholipase A2 complex with the hydrolysis products of platelet activating factor: equilibrium binding of fatty acid and lysophospholipid-ether at the active site may be mutually exclusive

We have solved the 1.55 A crystal structure of the anion-assisted dimer of porcine pancreatic group IB phospholipase A2 (PLA2), complexed with the products of hydrolysis of the substrate platelet activating factor. The dimer contains five coplanar phosphate anions bound at the contact surface between the two PLA2 subunits. This structure parallels a previously reported anion-assisted dimer that mimics the tetrahedral intermediate of PLA2 bound to a substrate interface [Pan, Y. H., et al. (2001) Biochemistry 40, 609-617]. The dimer structure has a molecule of the product acetate bound in subunit A and the other product 1-octadecyl-sn-glycero-3-phosphocholine (LPC-ether) to subunit B. Therefore, this structure is of the two individual product binary complexes and not of a ternary complex with both products in one active site of PLA2. Protein crystals with bound products were only obtained by cocrystallization starting from the initial substrate. In contrast, an alternate crystal form was obtained when PLA2 was cocrystallized with LPC-ether and succinate, and this crystal form did not contain bound products. The product bound structure has acetate positioned in the catalytic site of subunit A such that one of its oxygen atoms is located 3.5 A from the catalytic calcium. Likewise, a longer than typical Ca-to-Gly(32) carbonyl distance of 3.4 A results in a final Ca coordination that is four-coordinate and has distorted geometry. The other oxygen of acetate makes hydrogen bonds with N(delta)(1)-His(48), O(delta)(1)-Asp(49), and the catalytic assisting water (w7). In contrast, the glycerophosphocholine headgroup of LPC-ether in subunit B makes no contacts with calcium or with the catalytic residues His(48) or Asp(49). The tail of the LPC-ether is located near the active site pocket with the last nine carbons of the sn-1- acyl chain refined in two alternate conformations. The remaining atoms of the LPC-ether product have been modeled into the solvent channel but have their occupancies set to zero in the refined model due to disorder. Together, the crystallographic and equilibrium binding results with the two products show that the simultaneous binding of both the products in a single active site is not favored.     

Aspartate transcarbamoylase genes of Pseudomonas putida: requirement for an inactive dihydroorotase for assembly into the dodecameric holoenzyme.

The nucleotide sequences of the genes encoding the enzyme aspartate transcarbamoylase (ATCase) from Pseudomonas putida have been determined. Our results confirm that the P. putida ATCase is a dodecameric protein composed of two types of polypeptide chains translated coordinately from overlapping genes. The P. putida ATCase does not possess dissociable regulatory and catalytic functions but instead apparently contains the regulatory nucleotide binding site within a unique N-terminal extension of the pyrB-encoded subunit. The first gene, pyrB, is 1,005 bp long and encodes the 334-amino-acid, 36.4-kDa catalytic subunit of the enzyme. The second gene is 1,275 bp long and encodes a 424-residue polypeptide which bears significant homology to dihydroorotase (DHOase) from other organisms. Despite the homology of the overlapping gene to known DHOases, this 44.2-kDa polypeptide is not considered to be the functional product of the pyrC gene in P. putida, as DHOase activity is distinct from the ATCase complex. Moreover, the 44.2-kDa polypeptide lacks specific histidyl residues thought to be critical for DHOase enzymatic function. The pyrC-like gene (henceforth designated pyrC') does not complement Escherichia coli pyrC auxotrophs, while the cloned pyrB gene does complement pyrB auxotrophs. The proposed function for the vestigial DHOase is to maintain ATCase activity by conserving the dodecameric assembly of the native enzyme. This unique assembly of six active pyrB polypeptides coupled with six inactive pyrC' polypeptides has not been seen previously for ATCase but is reminiscent of the fused trifunctional CAD enzyme of eukaryotes.     

Recognition of ribosomal protein L11 by the protein trimethyltransferase PrmA

Bacterial ribosomal protein L11 is post-translationally trimethylated at multiple residues by a single methyltransferase, PrmA. Here, we describe four structures of PrmA from the extreme thermophile Thermus thermophilus. Two apo-PrmA structures at 1.59 and 2.3 A resolution and a third with bound cofactor S-adenosyl-L-methionine at 1.75 A each exhibit distinct relative positions of the substrate recognition and catalytic domains, revealing how PrmA can position the L11 substrate for multiple, consecutive side-chain methylation reactions. The fourth structure, the PrmA-L11 enzyme-substrate complex at 2.4 A resolution, illustrates the highly specific interaction of the N-terminal domain with its substrate and places Lys39 in the PrmA active site. The presence of a unique flexible loop in the cofactor-binding site suggests how exchange of AdoMet with the reaction product S-adenosyl-L-homocysteine can occur without necessitating the dissociation of PrmA from L11. Finally, the mode of interaction of PrmA with L11 explains its observed preference for L11 as substrate before its assembly into the 50S ribosomal subunit.     

X-ray structure of 5-aminolevulinic acid dehydratase from Escherichia coli complexed with the inhibitor levulinic acid at 2.0 A resolution

5-Aminolevulinic acid dehydratase (ALAD), an early enzyme of the tetrapyrrole biosynthesis pathway, catalyzes the dimerization of 5-aminolevulinic acid to form the pyrrole, porphobilinogen. ALAD from Escherichia coli is shown to form a homo-octameric structure with 422 symmetry in which each subunit adopts the TIM barrel fold with a 30-residue N-terminal arm. 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 lysine residues (195 and 247), one of which (Lys 247) forms a Schiff base link with the bound substrate analogue, levulinic acid. Of the two substrate binding sites (referred to as A and P), our analysis defines the residues forming the P-site, which is where the first ALA molecule to associate with the enzyme binds. The carboxyl group of the levulinic acid moiety forms hydrogen bonds with the side chains of Ser 273 and Tyr 312. In proximity to the levulinic acid is a zinc binding site formed by three cysteines (Cys 120, 122, and 130) and a solvent molecule. We infer that the second substrate binding site (or A-site) is located between the triple-cysteine zinc site and the bound levulinic acid moiety. Two invariant arginine residues in a loop covering the active site (Arg 205 and Arg 216) appear to be appropriately placed to bind the carboxylate of the A-site substrate. Another metal binding site, close to the active site flap, in which a putative zinc ion is coordinated by a carboxyl and five solvent molecules may account for the activating properties of magnesium ions.