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The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C5 precursors (such as ribose) during growth on other (non-C5) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PPi-PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PPi-PFKs of Clostridium thermosuccinogenes and Clostridium thermocellum indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of Escherichia coli, which does rely on transaldolase, had a very poor affinity for S7P. This indicates that the PPi-PFK of cellulolytic clostridia has evolved the use of S7P. We further show that C. thermosuccinogenes contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Last, we demonstrate that a second PFK of C. thermosuccinogenes that operates with ATP and GTP exhibits unusual kinetics toward F6P, as it appears to have an extremely high degree of cooperative binding, resulting in a virtual on/off switch for substrate concentrations near its K1/2 value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia.

Trehalose-6-phosphate synthase OtsA from streptomycetes is unusual in that it uses GDP-glucose as the donor substrate rather than the more commonly used UDP-glucose. We now confirm that OtsA from Streptomyces venezuelae has such a preference for GDP-glucose and can utilize ADP-glucose to some extent too. A crystal structure of the enzyme shows that it shares twin Rossmann-like domains with the UDP-glucose-specific OtsA from Escherichia coli. However, it is structurally more similar to Streptomyces hygroscopicus VldE, a GDP-valienol-dependent pseudoglycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost all identical in these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr, and Val) are similar in S. venezuelae OtsA (Asp, Ser, and Phe, respectively) but not conserved in E. coli OtsA (His, Leu, and Asp, respectively), providing a rationale for the purine base specificity of S. venezuelae OtsA. To establish which donor is used in vivo, we generated an otsA null mutant in S. venezuelae. The mutant had a cell density-dependent growth phenotype and accumulated galactose 1-phosphate, glucose 1-phosphate, and GDP-glucose when grown on galactose. To determine how the GDP-glucose is generated, we characterized three candidate GDP-glucose pyrophosphorylases. SVEN_3027 is a UDP-glucose pyrophosphorylase, SVEN_3972 is an unusual ITP-mannose pyrophosphorylase, and SVEN_2781 is a pyrophosphorylase that is capable of generating GDP-glucose as well as GDP-mannose. We have therefore established how S. venezuelae can make and utilize GDP-glucose in the biosynthesis of trehalose 6-phosphate. Fil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Miah, Farzana. John Innes Institute; Reino Unido Fil: Stevenson, Clare E. M.. John Innes Institute; Reino Unido Fil: Lawson, David M.. John Innes Institute; Reino Unido Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Bornemann, Stephen. John Innes Institute; Reino Unido

Background: Arabidopsis maltodextrin disproportionating enzyme 1 (AtDPE1) plays a key role in chloroplast starch degradation. Results: Six AtDPE1 structures define the active site and reveal mechanistically relevant conformations of both the enzyme and substrate. Conclusion: Substrates are captured through loop rearrangements; the subtle deployment of active site residues controls catalysis. Significance: A molecular level understanding of the complete disproportionation cycle of AtDPE1 is presented., The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whereas export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-α-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1), and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long, and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The “gate” is the most dynamic loop and appears to play a role in substrate capture, in particular in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms.

Oxalate decarboxylase (EC 4.1.1.2) catalyzes the conversion of oxalate to formate and carbon dioxide and utilizes dioxygen as a cofactor. By contrast, the evolutionarily related oxalate oxidase (EC 1.2.3.4) converts oxalate and dioxygen to carbon dioxide and hydrogen peroxide. Divergent free radical catalytic mechanisms have been proposed for these enzymes that involve the requirement of an active site proton donor in the decarboxylase but not the oxidase reaction. The oxidase possesses only one domain and manganese binding site per subunit, while the decarboxylase has two domains and two manganese sites per subunit. A structure of the decarboxylase together with a limited mutagenesis study has recently been interpreted as evidence that the C-terminal domain manganese binding site (site 2) is the catalytic site and that Glu-333 is the crucial proton donor (Anand, R., Dorrestein, P. C., Kinsland, C., Begley, T. P., and Ealick, S. E. (2002) Biochemistry 41, 7659-7669). The N-terminal binding site (site 1) of this structure is solvent-exposed (open) and lacks a suitable proton donor for the decarboxylase reaction. We report a new structure of the decarboxylase that shows a loop containing a 3(10) helix near site 1 in an alternative conformation. This loop adopts a "closed" conformation forming a lid covering the entrance to site 1. This conformational change brings Glu-162 close to the manganese ion, making it a new candidate for the crucial proton donor. Site-directed mutagenesis of equivalent residues in each domain provides evidence that Glu-162 performs this vital role and that the N-terminal domain is either the sole or the dominant catalytically active domain.

The optimal amino acid sequence of substrates for recognition by the cyclin-dependent kinases is well established as -Ser/Thr0-Pro+1-Lys+2-Lys+3-. The catalytic efficiency of CDK2-cyclin A is impaired 2000-, 10-, and 150-fold, when Pro+1, Lys+2, or Lys+3, respectively, is substituted with Ala in a short synthetic peptide substrate. Yet, in physiological substrates of both CDK2-cyclin A and CDK2-cyclin E, it is found that Lys+2, and, occasionally, both Lys+2 and Lys+3 together are replaced with suboptimal determinants. Such suboptimal phosphorylation site motifs are invariably associated with a distinct cyclin-binding (Cy) motif, which has been shown to compensate for otherwise poor catalysis. Here we have investigated the kinetic basis for substrate recognition by CDK2-cyclin A. In the optimal motif, Pro+1 serves to dramatically enhance both substrate binding affinity as well as the rate of chemical phosphotransfer, whereas Lys+2 and Lys+3 both serve to enhance mainly substrate binding. When linked to a suboptimal phosphorylation site sequence (Lys+2 → Pro) the Cy motif increases catalytic efficiency (kcat/Km) by increasing affinity without affecting turnover (kcat). When fused to the optimal sequence, however, catalytic efficiency is only minimally enhanced, because the resulting high substrate affinity impedes the rate of the phosphoryl transfer reaction. Our results provide kinetic insight into the basis for selecting suboptimal specificity determinants for the phosphorylation of cellular substrates.

Immunoscreening and DNA hybridization were used to isolate a 1.72-kilobase pair cDNA encoding cytosolic NADP(+)-dependent isocitrate dehydrogenase from a rat liver, lambda gt11 cDNA library. The identity of the cDNA was confirmed by comparison of the deduced amino acid sequence with sequences of peptides obtained from purified ovarian cytosolic isocitrate dehydrogenase. The 1.72-kilobase pair cDNA sequence translated into a protein of 414 amino acid residues with a molecular mass of 46,681 Da. The amino acid sequence contains a tripeptide (AKL) at the COOH terminus which represents a possible peroxisomal targeting sequence. The deduced amino acid sequence of the rat liver cytosolic isocitrate dehydrogenase showed 70 and 59% identity with sequences reported for NADP(+)-dependent isocitrate dehydrogenases from porcine mitochondria and yeast cytosol respectively. Northern blot analysis demonstrated a 13-fold increase in expression of cytosolic NADP(+)-dependent isocitrate dehydrogenase mRNA during the gonadotropin-induced development of the immature rat ovary. In comparative studies, the cytosolic and mitochondrial isocitrate dehydrogenase mRNAs were found to differ in size (2.2 and 1.8 kilobases, respectively) and to be differentially expressed in various tissues of the rat. Distinct digestion patterns were also obtained in Southern blot analysis of rat genomic DNA.

Achlya is a filamentous coenocytic fungus that grows only in the presence of Ca2+. The Ca2+ transport (uptake and binding) system of this organism was studied and shown to consist of two components: a low molecular weight Ca2+ binding glycopeptide present within the cell wall matrix and a Ca2+ uptake component associated with the cell membrane. Certain thiol group-reacting agents and cytokinin-active compounds enhanced Ca2+ binding and uptake, respectively. Ca2+ transport by intact cells showed pH dependence with optima at pH 6.5 and 8.5 and temperature dependence with maximal transport occurring at 40° (without activation) and 15° (with activation). Ca2+ transport proceeded by facilitated diffusion. The system is insensitive to metabolic inhibitors. Compounds with known cytokinin activity stimulated the release of Ca2+ bound to the cell wall glycopeptide, and these same compounds enhanced Ca2+ intake into osmotically shocked cells (lacking the glycopeptide). The half-saturation values for Ca2+ intake into cells and release from the glycopeptide are essentially identical.

Purified NAD-specified glutamic dehydrogenases from two fungi, Pythium debaryanum and Achlya sp. (1969), have been studied kinetically, and some of their physical properties elucidated. The enzymes are regulated by a multitude of allosteric effectors: NADP+, GTP, short chain acyl-CoA derivatives, P-enolpyruvate, and ATP as activators; citrate, AMP, and long chain CoA derivatives as inhibitors. The kinetic mechanism was determined for activated and non-activated enzymes. In both cases, an ordered binary-ternary mechanism prevailed. The order of substrate addition and product release is identical with the order found for all NADP-specific glutamic dehydrogenases although NADP+ is not a substrate of the enzyme. It is proposed that the enzyme may have evolved as a catalyst that was initially non-specific for both coenzymes and later became modified to accommodate NADP+ (or NADPH) as an allosteric activator. The enzymes are extremely heat labile. They denature rapidly above 37°. The activators and inhibitors fail to stabilize the proteins against heat denaturation. A novel effect of pH influence on the degree of activation and inhibition was observed. The enzymes are activated and inhibited best at the pH minima and least of all at the optimum pH. The relevance of these pH effects to the mitochondrial location of the enzymes is discussed.

A novel form of allosteric control in which NAD-specific glutamic dehydrogenases obtained from taxonomically related fungi are activated by NADP+, NADPH, and P-enolpyruvate has been elucidated. Citrate and isocitrate are equally effective as inhibitors of the enzymes. The enzymes are allosterically inhibited by their substrates, α-ketoglutarate and ammonia, in a highly cooperative manner. NADP+, NADPH, and P-enolpyruvate antagonize substrate inhibition. These activators also function as multivalent modulators in releasing the enzyme from inhibition by citrate. Control of the enzyme activity is unidirectionally oriented such that it is the reductive amination that is effectively controlled. The purified glutamic dehydrogenases have a molecular weight of 225,000 daltons. They are susceptible to inactivation by hydrodynamic shear in sucrose density gradients at alkaline pH levels. Inactivation by sedimentation can be prevented by bound activators. Inactivation and dissociation of the enzyme are related phenomena. Synthesis of the glutamic dehydrogenases can be repressed by addition of glucose to the growth medium. Glutamate acts as an inducer. NADP-specific isocitric dehydrogenase present in these fungi responds in the opposite manner to the metabolism of glucose and glutamate.

Guanylates (GTP, GDP, and GMP), short chain acyl-CoA derivatives; ATP, and ADP, were found to be allosteric activators of NAD-specific glutamic dehydrogenases isolated from a unique group of fungi, the Oomycetes. Long chain acyl-CoA derivatives and AMP were very strong inhibitors of the enzyme. Pythium glutamic dehydrogenase, in particular, was shown to be sensitive to control by energy charge. A total of five activators have now been discovered. These are NADP+, P-enolpyruvate, short chain acyl-CoA derivatives, GTP, and ATP. These activators functioned cumulatively to antagonize the effects of the inhibitors, citrate, AMP, and long chain acyl-CoA derivatives. The activators acted as unidirectional stimulants of the biosynthetic reaction, while some of them inhibited the catabolic reaction unidirectionally. These glutamic dehydrogenases are genetically controlled. They are subjected to marked catabolic repression and inducible by glutamate. The substrates (ammonia and α-ketoglutarate) of the biosynthetic reaction are allosteric inhibitors. The activators modulated the enzyme against allosteric inhibition by its substrates. This multivalent control has been explained as a mechanism by which the enzyme effects its biosynthetic and cataboilc roles in amphibolic reactions of the citric acid cycle. A remarkable correlation has been made between lysine biosynthesis and allosteric control mechanisms among members of the Phycomycetes. The distribution of the two pathways of lysine biosynthesis among these simple fungi parallels the only two different forms of allosteric controls of NAD-specific glutamic dehydrogenases elucidated for all major orders of the Phycomycetes studied to date.