Moritella cold-active dihydrofolate reductase: are there natural limits to optimization of catalytic efficiency at low temperature?

Adapting metabolic enzymes of microorganisms to low temperature environments may require a difficult compromise between velocity and affinity. We have investigated catalytic efficiency in a key metabolic enzyme (dihydrofolate reductase) of Moritella profunda sp. nov., a strictly psychrophilic bacterium with a maximal growth rate at 2 degrees C or less. The enzyme is monomeric (Mr=18,291), 55% identical to its Escherichia coli counterpart, and displays Tm and denaturation enthalpy changes much lower than E. coli and Thermotoga maritima homologues. Its stability curve indicates a maximum stability above the temperature range of the organism, and predicts cold denaturation below 0 degrees C. At mesophilic temperatures the apparent Km value for dihydrofolate is 50- to 80-fold higher than for E. coli, Lactobacillus casei, and T. maritima dihydrofolate reductases, whereas the apparent Km value for NADPH, though higher, remains in the same order of magnitude. At 5 degrees C these values are not significantly modified. The enzyme is also much less sensitive than its E. coli counterpart to the inhibitors methotrexate and trimethoprim. The catalytic efficiency (kcat/Km) with respect to dihydrofolate is thus much lower than in the other three bacteria. The higher affinity for NADPH could have been maintained by selection since NADPH assists the release of the product tetrahydrofolate. Dihydrofolate reductase adaptation to low temperature thus appears to have entailed a pronounced trade-off between affinity and catalytic velocity. The kinetic features of this psychrophilic protein suggest that enzyme adaptation to low temperature may be constrained by natural limits to optimization of catalytic efficiency.     

Metabolic enzymes from psychrophilic bacteria: challenge of adaptation to low temperatures in ornithine carbamoyltransferase from Moritella abyssi

The enzyme ornithine carbamoyltransferase (OTCase) of Moritella abyssi (OTCase(Mab)), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCase(Mab) displays maximal activity at rather low temperatures (23 to 25 degrees C) compared to other cold-active enzymes and is much less thermoresistant than its homologues from Escherichia coli or thermophilic procaryotes. In vitro the enzyme is in equilibrium between a trimeric state and a dodecameric, more stable state. The melting point and denaturation enthalpy changes for the two forms are considerably lower than the corresponding values for the dodecameric Pyrococcus furiosus OTCase and for a thermolabile trimeric mutant thereof. OTCase(Mab) displays higher K(m) values for ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue delta-N-phosphonoacetyl-L-ornithine (PALO). OTCase(Mab) differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high K(m) values and the lower sensitivity to PALO. The K(m) for ornithine, however, is substantially lower at low temperatures. A survey of the catalytic efficiencies (k(cat)/K(m)) of OTCases adapted to different temperatures showed that OTCase(Mab) activity remains suboptimal at low temperature despite the 4.5-fold decrease in the K(m) value for ornithine observed when the temperature is brought from 20 to 5 degrees C. OTCase(Mab) adaptation to cold indicates a trade-off between affinity and catalytic velocity, suggesting that optimization of key metabolic enzymes at low temperatures may be constrained by natural limits.     

Protein kinase A blocks Raf-1 activity by stimulating 14-3-3 binding and blocking Raf-1 interaction with Ras

Cyclic AMP (cAMP) blocks Raf-1 activation by stimulating its phosphorylation on serine 43 (Ser43), serine 233 (Ser233), and serine 259 (Ser259). We show here that phosphorylation of all three sites blocks Raf-1 binding to Ras.GTP in vivo and that cAMP stimulates binding of 14-3-3 proteins to Ser233 and Ser259. We also show that Raf-1 and protein kinase A (PKA) form a complex in vivo that is disrupted by cAMP and that ablation of PKA by use of small interfering RNA blocks phosphorylation by cAMP. The ability of PKA to block Raf-1 activation is ablated by the PKA inhibitor H89. These studies suggest that Raf-1 and cAMP form a signaling complex in cells. Upon activation of PKA, Raf-1 is phosphorylated and 14-3-3 binds, blocking Raf-1 recruitment to the plasma membrane and preventing its activation.     

A recombinant β-O-glucosidase from Caldocellum saccharolyticum to hydrolyse desulfo-glucosinolates

Glucosinolates, natural compounds found in Brassicaceae, can easily be transformed into desulfo-glucosinolates by action of Helix pomatia sulfatase. The recombinant -O-glucosidase from Caldocellum saccharolyticum does not catalyse glucosinolate degradation but can hydrolyse desulfo-glucosinolates (thio-d-glucosidic substrates) to produce the corresponding pure nitriles, including valuable homochiral representatives.     

The mu-selective heptapeptide opioid dermorphin has two conformations around Phe3 psi with no head-to-tail interaction. A quantitative 2-D NMR and molecular modeling analysis

The mu opioid heptapeptide Dermorphin (DRM) is under 70 % of trans forms for the Tyr(5)-Pro(6) peptide bond in solution (CDCl(3)/DMSO-d(6) 1/1 vol/vol). Variations of NOE integrals at 5 temperatures show apparent correlation times of 0.8 to 0.9 ns (at 280 K) in that mixed solvent. Four NOE between non-adjacent residues reveal a large population of folded structures. However, in trans DRM, 4 adjacent NOE Phe(3)/Gly(4) can only be explained by an equilibrium between folded (psi(3) > 0) and extended (psi(3) > 0) conformations. Simulated annealing modeling gave about 60% (psi(3) > 0) and 40% (psi(3) > 0) of these conformer populations. Trans DRM study and previous studies on the heptapeptide opioids, dermenkephalin (DREK) and deltorphin-I (delta selective), and DREK(1-4)-DRM(5-7) hybrid (mu selective), show in folded structures more backbone bending of the first 4 residues in the mu opioids than in the delta peptides. Also, the main difference between mu- and delta-opioid peptides is a large fraction of extended conformations in mu heptapeptides. Either bending of the N-terminus, or extension of the C-terminal part in mu-opioid heptapeptides prevent the head-to-tail interactions which allow delta-opioid peptides to bind selectively to the delta-opioid receptor.     

Selective inhibition of dipeptidyl peptidase I,not caspases,prevents the partial processing of procaspase-3 in CD3-activated human CD8(+) T lymphocytes

Activation of primary human T cells by anti-CD3 and interleukin-2 resulted in partial processing of procaspase-3 in activated nonapoptotic (Delta Psi(m)high) CD8(+) T cells but not in CD4(+) T cells. Apical caspases-8 and -9 were not activated, and Bid was not processed to truncated Bid. Boc-D.fmk, a broad spectrum caspase inhibitor, did not prevent this process, whereas GF.dmk, a selective inhibitor of dipeptidyl peptidase I, was effective. Dipeptidyl peptidase I is required for the activation of granule-associated serine proteases. It is enriched in the cytolytic granules of cytotoxic lymphocytes, where it promotes the proteolytic activation of progranzymes A and B. Inhibition of granzyme B (GrB)-like serine proteases by Z-AAD.cmk prevented partial processing of procapase-3, whereas inhibition of GrA activity by D-FPR.cmk had no effect. Specific inhibitors of other lysosomal proteases such as cathepsins B, L, and D did not interfere in this event. Patients with Chediak-Higashi syndrome or with perforin deficiency also displayed partial processing of procaspase-3, excluding the involvement of granule exocytosis for the delivery of the serine protease in cause. The p20/p12 processing pattern of procaspase-3 in our model points to GrB, the sole serine protease with caspase activity. Small amounts of GrB were indeed exported from cytolytic granules to the cytosol of a significant fraction of GrB-positive cells.     

A covarion-based method for detecting molecular adaptation: application to the evolution of primate mitochondrial genomes

A new method for detecting site-specific variation of evolutionary rate (the so-called covarion process) from protein sequence data is proposed. It involves comparing the maximum-likelihood estimates of the replacement rate of an amino acid site in distinct subtrees of a large tree. This approach allows detection of covarion at the gene or the amino acid levels. The method is applied to mammalian-mitochondrial-protein sequences. Significant covarion-like evolution is found in the (simian) primate lineage: some amino acid positions are fast-evolving (i.e. unconstrained) in non-primate mammals but slow-evolving (i.e. highly constrained) in primates, and some show the opposite pattern. Our results indicate that the mitochondrial genome of primates reached a new peak of the adaptive landscape through positive selection.     

Targeted stimulation of meiotic recombination

Meiotic recombination in Saccharomyces cerevisiae is initiated by programmed DNA double-strand breaks (DSBs), a process that requires the Spo11 protein. DSBs usually occur in intergenic regions that display open chromatin accessibility, but other determinants that control their frequencies and non-random chromosomal distribution remain obscure. We report that a Spo11 construct bearing the Gal4 DNA binding domain not only rescues spo11Delta spore inviability and catalyzes DSB formation at natural sites but also strongly stimulates DSB formation near Gal4 binding sites. At GAL2, a naturally DSB-cold locus, Gal4BD-Spo11 creates a recombinational hotspot that depends on all the other DSB gene functions, showing that the targeting of Spo11 to a specific site is sufficient to stimulate meiotic recombination that is under normal physiological control.