Mechanism of Galpha i-mediated inhibition of type V adenylyl cyclase

The topology of mammalian adenylyl cyclase reveals an integral membrane protein composed of an alternating series of membrane and cytoplasmic domains (C1 and C2). The stimulatory G protein, Galpha(s), binds within a cleft in the C2 domain of adenylyl cyclase while Galpha(i) binds within the opposite cleft in the C1 domain. The mechanism of these two regulators also appears to be in opposition. Activation of adenylyl cyclase by Galpha(s) or forskolin results in a 100-fold increase in the apparent affinity of the two domains for one another. We show herein that Galpha(i) reduces C1/C2 domain interaction and thus formation of the adenylyl cyclase catalytic site. Mutants that increase the affinity of C1 for C2 decrease the ability of Galpha(i) to inhibit the enzyme. In addition, Galpha(i) can influence binding of molecules to the catalytic site, which resides at the C1/C2 interface. Adenylyl cyclase can bind substrate analogs in the presence of Galpha(i) but cannot simultaneously bind Galpha(i) and transition state analogs such as 2'd3'-AMP. Galpha(i) also cannot inhibit the membrane-bound enzyme in the presence of manganese, which increases the affinity of adenylyl cyclase for ATP and substrate analogs. Thus homologous G protein alpha-subunits promote bidirectional regulation at the domain interface of the pseudosymmetrical adenylyl cyclase enzyme.     

Molecular details of cAMP generation in mammalian cells: a tale of two systems

The second messenger cAMP has been extensively studied for half a century, but the plethora of regulatory mechanisms controlling cAMP synthesis in mammalian cells is just beginning to be revealed. In mammalian cells, cAMP is produced by two evolutionary related families of adenylyl cyclases, soluble adenylyl cyclases (sAC) and transmembrane adenylyl cyclases (tmAC). These two enzyme families serve distinct physiological functions. They share a conserved overall architecture in their catalytic domains and a common catalytic mechanism, but they differ in their sub-cellular localizations and responses to various regulators. The major regulators of tmACs are heterotrimeric G proteins, which transduce extracellular signals via G protein-coupled receptors. sAC enzymes, in contrast, are regulated by the intracellular signaling molecules bicarbonate and calcium. Here, we discuss and compare the biochemical, structural and regulatory characteristics of the two mammalian AC families. This comparison reveals the mechanisms underlying their different properties but also illustrates many unifying themes for these evolutionary related signaling enzymes.     

Importance of domain closure for the catalysis and regulation of Escherichia coli aspartate transcarbamoylase

Two hybrid versions of Escherichia coli aspartate transcarbamoylase were studied to determine the influence of domain closure on the homotropic and heterotropic properties of the enzyme. Each hybrid holoenzyme had one wild-type and one inactive catalytic subunit. In the first case the inactive catalytic subunit had Arg-54 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 17,000-fold reduction in activity, no loss in substrate affinity, and an R state structurally identical to that of the wild-type enzyme. In the second case, the inactive catalytic subunit had Arg-105 replaced by alanine. The holoenzyme with this mutation in all six catalytic chains exhibits a 1,100-fold reduction in activity, substantial loss in substrate affinity, and loss of the ability to be converted to the R state. Thus, the R54A substitution results in a holoenzyme that can undergo closure of the catalytic chain domains to form the high activity, high affinity active site and to undergo the allosteric transition, whereas the R105A substitution results in a holoenzyme that can neither undergo domain closure nor the allosteric transition. The hybrid holoenzyme with one wild-type and one R54A catalytic subunit exhibited the same maximal velocity per active site as the wild-type holoenzyme, reduced cooperativity, and normal heterotropic interactions. The hybrid with one wild-type and one R105A catalytic subunit exhibited significantly reduced maximal velocity per active site as compared with the wild-type holoenzyme, reduced cooperativity, and substantially reduced heterotropic interactions. Small angle x-ray scattered was used to verify that the R105A-containing hybrid could attain an R state structure. These results indicate the global nature of the conformational changes associated with the allosteric transition in the enzyme. If one catalytic subunit cannot undergo domain closure to create the active sites, then the entire molecule cannot attain the high activity, high activity R state.     

A defined subset of adenylyl cyclases is regulated by bicarbonate ion

The molecular basis by which organisms detect and respond to fluctuations in inorganic carbon is not known. The cyaB1 gene of the cyanobacterium Anabaena sp. PCC7120 codes for a multidomain protein with a C-terminal class III adenylyl cyclase catalyst that was specifically stimulated by bicarbonate ion (EC50 9.6 mm). Bicarbonate lowered substrate affinity but increased reaction velocity. A point mutation in the active site (Lys-646) reduced activity by 95% and was refractory to bicarbonate activation. We propose that Lys-646 specifically coordinates bicarbonate in the active site in conjunction with an aspartate to threonine polymorphism (Thr-721) conserved in class III adenylyl cyclases from diverse eukaryotes and prokaryotes. Using recombinant proteins we demonstrated that adenylyl cyclases that contain the active site threonine (cyaB of Stigmatella aurantiaca and Rv1319c of Mycobacterium tuberculosis) are bicarbonate-responsive, whereas adenylyl cyclases with a corresponding aspartate (Rv1264 of Mycobacterium) are bicarbonate-insensitive. Large numbers of class III adenylyl cyclases may therefore be activated by bicarbonate. This represents a novel mechanism by which diverse organisms can detect bicarbonate ion.     

Crystal Structures Capture Three States in the Catalytic Cycle of a Pyridoxal Phosphate (PLP) Synthase

PLP synthase (PLPS) is a remarkable single-enzyme biosynthetic pathway that produces pyridoxal 5′-phosphate (PLP) from glutamine, ribose 5-phosphate, and glyceraldehyde 3-phosphate. The intact enzyme includes 12 synthase and 12 glutaminase subunits. PLP synthesis occurs in the synthase active site by a complicated mechanism involving at least two covalent intermediates at a catalytic lysine. The first intermediate forms with ribose 5-phosphate. The glutaminase subunit is a glutamine amidotransferase that hydrolyzes glutamine and channels ammonia to the synthase active site. Ammonia attack on the first covalent intermediate forms the second intermediate. Glyceraldehyde 3-phosphate reacts with the second intermediate to form PLP. To investigate the mechanism of the synthase subunit, crystal structures were obtained for three intermediate states of the Geobacillus stearothermophilus intact PLPS or its synthase subunit. The structures capture the synthase active site at three distinct steps in its complicated catalytic cycle, provide insights into the elusive mechanism, and illustrate the coordinated motions within the synthase subunit that separate the catalytic states. In the intact PLPS with a Michaelis-like intermediate in the glutaminase active site, the first covalent intermediate of the synthase is fully sequestered within the enzyme by the ordering of a generally disordered 20-residue C-terminal tail. Following addition of ammonia, the synthase active site opens and admits the Lys-149 side chain, which participates in formation of the second intermediate and PLP. Roles are identified for conserved Asp-24 in the formation of the first intermediate and for conserved Arg-147 in the conversion of the first to the second intermediate.     

Kinetic properties of "soluble" adenylyl cyclase. Synergism between calcium and bicarbonate

"Soluble" adenylyl cyclase (sAC) is a widely expressed source of cAMP in mammalian cells that is evolutionarily, structurally, and biochemically distinct from the G protein-responsive transmembrane adenylyl cyclases. In contrast to transmembrane adenylyl cyclases, sAC is insensitive to heterotrimeric G protein regulation and forskolin stimulation and is uniquely modulated by bicarbonate ions. Here we present the first report detailing kinetic analysis and biochemical properties of purified recombinant sAC. We confirm that bicarbonate regulation is conserved among mammalian sAC orthologs and demonstrate that bicarbonate stimulation is consistent with an increase in the V(max) of the enzyme with little effect on the apparent K(m) for substrate, ATP-Mg(2+). Bicarbonate can further increase sAC activity by relieving substrate inhibition. We also identify calcium as a direct modulator of sAC activity. In contrast to bicarbonate, calcium stimulates sAC activity by decreasing its apparent K(m) for ATP-Mg(2+). Because of their different mechanisms, calcium and bicarbonate synergistically activate sAC; therefore, small changes of either calcium or bicarbonate will lead to significant changes in cellular cAMP levels.     

The crystal structure of a novel bacterial adenylyltransferase reveals half of sites reactivity

Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in bacteria that catalyses a rate-limiting step in coenzyme A (CoA) biosynthesis, by transferring an adenylyl group from ATP to 4'-phosphopantetheine, yielding dephospho-CoA (dPCoA). Each phosphopantetheine adenylyltransferase (PPAT) subunit displays a dinucleotide-binding fold that is structurally similar to that in class I aminoacyl-tRNA synthetases. Superposition of bound adenylyl moieties from dPCoA in PPAT and ATP in aminoacyl-tRNA synthetases suggests nucleophilic attack by the 4'-phosphopantetheine on the alpha-phosphate of ATP. The proposed catalytic mechanism implicates transition state stabilization by PPAT without involving functional groups of the enzyme in a chemical sense in the reaction. The crystal structure of the enzyme from Escherichia coli in complex with dPCoA shows that binding at one site causes a vice-like movement of active site residues lining the active site surface. The mode of enzyme product formation is highly concerted, with only one trimer of the PPAT hexamer showing evidence of dPCoA binding. The homologous active site attachment of ATP and the structural distribution of predicted sequence-binding motifs in PPAT classify the enzyme as belonging to the nucleotidyltransferase superfamily.     

Structure-based computational study of the catalytic and inhibition mechanisms of urease

The viability of different mechanisms of catalysis and inhibition of the nickel-containing enzyme urease was explored using the available high-resolution structures of the enzyme isolated from Bacillus pasteurii in the native form and inhibited with several substrates. The structures and charge distribution of urea, its catalytic transition state, and three enzyme inhibitors were calculated using ab initio and density functional theory methods. The DOCK program suite was employed to determine families of structures of urease complexes characterized by docking energy scores indicative of their relative stability according to steric and electrostatic criteria. Adjustment of the parameters used by DOCK, in order to account for the presence of the metal ion in the active site, resulted in the calculation of best energy structures for the nickel-bound inhibitors beta-mercaptoethanol, acetohydroxamic acid, and diamidophosphoric acid. These calculated structures are in good agreement with the experimentally determined structures, and provide hints on the reactivity and mobility of the inhibitors in the active site. The same docking protocol was applied to the substrate urea and its catalytic transition state, in order to shed light onto the possible catalytic steps occurring at the binuclear nickel active site. These calculations suggest that the most viable pathway for urea hydrolysis involve a nucleophilic attack by the bridging, and not the terminal, nickel-bound hydroxide onto a urea molecule, with active site residues playing important roles in orienting and activating the substrate, and stabilizing the catalytic transition state.     

Studies of Glu-416 Variants of β-Galactosidase (<Emphasis Type="Italic">E. coli</Emphasis>) Show that the Active Site Mg<Superscript>2+</Superscript> is Not Important for Structure and Indicate that the Main Role of Mg<Superscript>2+</Superscript> is to Mediate Optimization of Active Site Chemistry

Variants of β-galactosidase with Valine and with Glutamine replacing Glutamate-416 did not have a Mg²⁺ bound at the active site even at high Mg²⁺ concentrations (200 mM). They had low catalytic activity and the pH profiles were very different from those of the native enzyme. In addition, substrates, substrate analogs, transition state analogs and galactose bound very poorly. However, the orientation and conformation of the Mg²⁺ ligands (residues 416, 418, and 461) as well as the B-factors of these three side chains did not change significantly. The structures, conformations and B-factors of other active site residues were also essentially unchanged. These studies show that the active site Mg²⁺ is not necessary for structure and is, therefore, mainly important for modulating the chemistry and mediating the interactions between the active site components.     

Glucose and GLP-1 stimulate cAMP production via distinct adenylyl cyclases in INS-1E insulinoma cells

In β cells, both glucose and hormones, such as GLP-1, stimulate production of the second messenger cAMP, but glucose and GLP-1 elicit distinct cellular responses. We now show in INS-1E insulinoma cells that glucose and GLP-1 produce cAMP with distinct kinetics via different adenylyl cyclases. GLP-1 induces a rapid cAMP signal mediated by G protein–responsive transmembrane adenylyl cyclases (tmAC). In contrast, glucose elicits a delayed cAMP rise mediated by bicarbonate, calcium, and ATP-sensitive soluble adenylyl cyclase (sAC). This glucose-induced, sAC-dependent cAMP rise is dependent upon calcium influx and is responsible for the glucose-induced activation of the mitogen-activated protein kinase (ERK1/2) pathway. These results demonstrate that sAC-generated and tmAC-generated cAMP define distinct signaling cascades.     

On the mechanism of biological methane formation: structural evidence for conformational changes in methyl-coenzyme M reductase upon substrate binding

Methyl-coenzyme M reductase (MCR) catalyzes the final reaction of the energy conserving pathway of methanogenic archaea in which methylcoenzyme M and coenzyme B are converted to methane and the heterodisulfide CoM-S-S-CoB. It operates under strictly anaerobic conditions and contains the nickel porphinoid F430 which is present in the nickel (I) oxidation state in the active enzyme. The known crystal structures of the inactive nickel (II) enzyme in complex with coenzyme M and coenzyme B (MCR-ox1-silent) and in complex with the heterodisulfide CoM-S-S-CoB (MCR-silent) were now refined at 1.16 A and 1.8 A resolution, respectively. The atomic resolution structure of MCR-ox1-silent describes the exact geometry of the cofactor F430, of the active site residues and of the modified amino acid residues. Moreover, the observation of 18 Mg2+ and 9 Na+ ions at the protein surface of the 300 kDa enzyme specifies typical constituents of binding sites for either ion. The MCR-silent and MCR-ox1-silent structures differed in the occupancy of bound water molecules near the active site indicating that a water chain is involved in the replenishment of the active site with water molecules. The structure of the novel enzyme state MCR-red1-silent at 1.8 A resolution revealed an active site only partially occupied by coenzyme M and coenzyme B. Increased flexibility and distinct alternate conformations were observed near the active site and the substrate channel. The electron density of the MCR-red1-silent state aerobically co-crystallized with coenzyme M displayed a fully occupied coenzyme M-binding site with no alternate conformations. Therefore, the structure was very similar to the MCR-ox1-silent state. As a consequence, the binding of coenzyme M induced specific conformational changes that postulate a molecular mechanism by which the enzyme ensures that methylcoenzyme M enters the substrate channel prior to coenzyme B as required by the active-site geometry. The three different enzymatically inactive enzyme states are discussed with respect to their enzymatically active precursors and with respect to the catalytic mechanism.     

An intermediate step in the recognition of tRNA(Asp) by aspartyl-tRNA synthetase

The crystal structures of aspartyl-tRNA synthetase (AspRS) from Thermus thermophilus, a prokaryotic class IIb enzyme, complexed with tRNA(Asp) from either T. thermophilus or Escherichia coli reveal a potential intermediate of the recognition process. The tRNA is positioned on the enzyme such that it cannot be aminoacylated but adopts an overall conformation similar to that observed in active complexes. While the anticodon loop binds to the N-terminal domain of the enzyme in a manner similar to that of the related active complexes, its aminoacyl acceptor arm remains at the entrance of the active site, stabilized in its intermediate conformational state by non-specific interactions with the insertion and catalytic domains. The thermophilic nature of the enzyme, which manifests itself in a very low kinetic efficiency at 17 degrees C, the temperature at which the crystals were grown, is in agreement with the relative stability of this non-productive conformational state. Based on these data, a pathway for tRNA binding and recognition is proposed.     

Insights into the mechanism of type I dehydroquinate dehydratases from structures of reaction intermediates

The biosynthetic shikimate pathway consists of seven enzymes that catalyze sequential reactions to generate chorismate, a critical branch point in the synthesis of the aromatic amino acids. The third enzyme in the pathway, dehydroquinate dehydratase (DHQD), catalyzes the dehydration of 3-dehydroquinate to 3-dehydroshikimate. We present three crystal structures of the type I DHQD from the intestinal pathogens Clostridium difficile and Salmonella enterica. Structures of the enzyme with substrate and covalent pre- and post-dehydration reaction intermediates provide snapshots of successive steps along the type I DHQD-catalyzed reaction coordinate. These structures reveal that the position of the substrate within the active site does not appreciably change upon Schiff base formation. The intermediate state structures reveal a reaction state-dependent behavior of His-143 in which the residue adopts a conformation proximal to the site of catalytic dehydration only when the leaving group is present. We speculate that His-143 is likely to assume differing catalytic roles in each of its observed conformations. One conformation of His-143 positions the residue for the formation/hydrolysis of the covalent Schiff base intermediates, whereas the other conformation positions the residue for a role in the catalytic dehydration event. The fact that the shikimate pathway is absent from humans makes the enzymes of the pathway potential targets for the development of non-toxic antimicrobials. The structures and mechanistic insight presented here may inform the design of type I DHQD enzyme inhibitors.     

Structural basis for the function of pyridoxine 5'-phosphate synthase

BACKGROUND: Pyridoxal 5'-phosphate is the active form of vitamin B(6) that acts as an essential, ubiquitous coenzyme in amino acid metabolism. In Escherichia coli, the pathway of the de novo biosynthesis of vitamin B(6) results in the formation of pyridoxine 5'-phosphate (PNP), which can be regarded as the first synthesized B(6) vitamer. PNP synthase (commonly referred to as PdxJ) is a homooctameric enzyme that catalyzes the final step in this pathway, a complex intramolecular condensation reaction between 1-deoxy-D-xylulose-5'-phosphate and 1-amino-acetone-3-phosphate. RESULTS: The crystal structure of E. coli PNP synthase was solved by single isomorphous replacement with anomalous scattering and refined at a resolution of 2.0 A. The monomer of PNP synthase consists of one compact domain that adopts the abundant TIM barrel fold. Intersubunit contacts are mediated by three additional helices, respective to the classical TIM barrel helices, generating a tetramer of symmetric dimers with 422 symmetry. In the shared active sites of the active dimers, Arg20 is directly involved in substrate binding of the partner monomer. Furthermore, the structure of PNP synthase with its physiological products, PNP and P(i), was determined at 2.3 A resolution, which provides insight into the dynamic action of the enzyme and allows us to identify amino acids critical for enzymatic function. CONCLUSION: The high-resolution structures of the free enzyme and the enzyme-product complex of E. coli PNP synthase suggest essentials of the enzymatic mechanism. The main catalytic features are active site closure upon substrate binding by rearrangement of one C-terminal loop of the TIM barrel, charge-charge stabilization of the protonated Schiff-base intermediate, the presence of two phosphate binding sites, and a water channel that penetrates the beta barrel and allows the release of water molecules in the closed state. All related PNP synthases are predicted to fold into a similar TIM barrel pattern and have comparable active site architecture. Thus, a common mechanism can be anticipated.     

Syntheses and structures of M(L)(X)BPh4 complexes {M=Co(II), Zn(II); L=cis-1,3,5-tris[3-(2-furyl)prop-2-enylideneamino]cyclohexane, X=OAc, NO3}: structural models of the active site of carbonic anhydrase

The metal coordination geometries in the structures of the zinc(II) and cobalt(II) complexes of the ligand cis-1,3,5-tris[3-(2-furyl)prop-2-enylideneamino]cyclohexane (fr-protach) and with the anions nitrate and acetate are structural models for the active site of carbonic anhydrase. The acetate structures show a striking structural correlation with the metal coordination environments in the known bicarbonate forms of the enzyme. Such structures provide a basis for understanding the marked effect of different metal substitution on the catalytic rate of the enzyme.     

Structure of the unliganded cAMP-dependent protein kinase catalytic subunit from Saccharomyces cerevisiae

The structure of TPK1delta, a truncated variant of the cAMP-dependent protein kinase catalytic subunit from Saccharomyces cerevisiae, was determined in an unliganded state at 2.8 A resolution and refined to a crystallographic R-factor of 19.4%. Comparison of this structure to that of its fully liganded mammalian homolog revealed a highly conserved protein fold comprised of two globular lobes. Within each lobe, root mean square deviations in Calpha positions averaged approximately equals 0.9 A. In addition, a phosphothreonine residue was found in the C-terminal domain of each enzyme. Further comparison of the two structures suggests that a trio of conformational changes accompanies ligand-binding. The first consists of a 14.7 degrees rigid-body rotation of one lobe relative to the other and results in closure of the active site cleft. The second affects only the glycine-rich nucleotide binding loop, which moves approximately equals 3 A to further close the active site and traps the nucleotide substrate. The third is localized to a C-terminal segment that makes direct contact with ligands and the ligand-binding cleft. In addition to resolving the conformation of unliganded enzyme, the model shows that the salient features of the cAMP-dependent protein kinase are conserved over long evolutionary distances.     

X-ray structure of prephenate dehydratase from Streptococcus mutans

Prephenate dehydratase is a key enzyme of the biosynthesis of L-phenylalanine in the organisms that utilize shikimate pathway. Since this enzymatic pathway does not exist in mammals, prephenate dehydratase can provide a new drug targets for antibiotics or herbicide. Prephenate dehydratase is an allosteric enzyme regulated by its end product. The enzyme composed of two domains, catalytic PDT domain located near the N-terminal and regulatory ACT domain located near the C-terminal. The allosteric enzyme is suggested to have two different conformations. When the regulatory molecule, phenylalanine, is not bound to its ACT domain, the catalytic site of PDT domain maintain open (active) state conformation as Sa-PDT structure. And the open state of its catalytic site become closed (allosterically inhibited) state if the regulatory molecule is bound to its ACT domain as Ct-PDT structure. However, the X-ray structure of prephenate dehydratase from Streptococcus mutans (Sm-PDT) shows that the catalytic site of Sm-PDT has closed state conformation without phenylalanine molecule bound to its regulatory site. The structure suggests a possibility that the binding of phenylalanine in its regulatory site may not be the only prerequisite for the closed state conformation of Sm-PDT.     

The crystal structures of phosphopantetheine adenylyltransferase with bound substrates reveal the enzyme's catalytic mechanism.

Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in the coenzyme A pathway that catalyzes the reversible transfer of an adenylyl group from ATP to 4'-phosphopantetheine (Ppant) in the presence of magnesium. To investigate the reaction mechanism, the high-resolution crystal structures of the Escherichia coli PPAT have been determined in the presence of either ATP or Ppant. Structural details of the catalytic center revealed specific roles for individual amino acid residues involved in substrate binding and catalysis. The side-chain of His18 stabilizes the expected pentacovalent intermediate, whereas the side-chains of Thr10 and Lys42 orient the nucleophile for an in-line displacement mechanism. The binding site for the manganese ion that interacts with the phosphate groups of the nucleotide has also been identified. Within the PPAT hexamer, one trimer is in its substrate-free state, whereas the other is in a substrate-bound state.     

Catalytic mechanism of the cyclohydrolase activity of human aminoimidazole carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase

The bifunctional enzyme aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is responsible for catalysis of the last two steps in the de novo purine pathway. Using recently determined crystal structures of ATIC as a guide, four candidate residues, Lys66, Tyr104, Asp125, and Lys137, were identified for site-directed mutagenesis to study the cyclohydrolase activity of this bifunctional enzyme. Steady-state kinetic experiments on these mutants have shown that none of these residues are absolutely required for catalytic activity; however, they strongly influence the efficiency of the reaction. Since the FAICAR binding site is made up mostly of backbone interactions with highly conserved residues, we postulate that these conserved interactions orient FAICAR in the active site to favor the intramolecular ring closure reaction and that this reaction may be catalyzed by an orbital steering mechanism. Furthermore, it was shown that Lys137 is responsible for the increase in cyclohydrolase activity for dimeric ATIC, which was reported previously by our laboratory. From the experiments presented here, a catalytic mechanism for the cyclohydrolase activity is postulated.