Conformational forces affecting the folding pathways of dendrotoxins I and K from black mamba venom

The conformations of the major intermediates trapped during the folding of dendrotoxins I and K from venom of black mamba snakes, have been investigated by circular-dichroism spectroscopy. Local alterations to the native, folded conformations are observed in toxins I and K and in a protein of similar sequence, bovine pancreatic trypsin inhibitor. The inability of intermediates (30-51, 14-38) to complete refolding by forming directly the 5-55 disulphide bond is explained. The following observations on the role of secondary structure in the folding of the three proteins are of interest. 1. It is not necessary for the three proteins to acquire elements of secondary structure at the same stage of folding in order to attain similar three-dimensional conformations. 2. The stability of the final folded state is not directly correlated to an early appearance of secondary structure. 3. The degree of secondary structure already present in intermediates (30-51) seems to determine the pathway of refolding preferred by the corresponding protein.     

Biosynthesis of the corrin macrocycle of coenzyme B12 in Pseudomonas denitrificans.

Studies with cell-free protein preparations from a series of recombinant strains of Pseudomonas denitrificans demonstrated that precorrin-3 is converted into a further trimethylated intermediate, named precorrin-3B, along the pathway to coenzyme B12. It was then shown that the part of the pathway from precorrin-3 (called precorrin-3A hereafter) to precorrin-6x involves three intermediates, precorrin-3B, precorrin-4, and precorrin-5. Precorrin-3B was isolated in its native (reduced) as well as its oxidized (factor-IIIB) states, and precorrin-4 was isolated in its oxidized form only (factor-IV). Both factors were in vitro precursors of precorrin-6x. The synthesis of precorrin-6x from precorrin-3A was shown to be catalyzed by four enzymes, CobG, CobJ, CobM, and CobF, intervening in this order. They were purified to homogeneity. CobG, which converts precorrin-3A to precorrin-3B, was found to be an iron-sulfur protein responsible for the oxidation known to occur between precorrin-3A and precorrin-6x, and CobJ, CobM, and CobF are the C-17, C-11, and C-1 methylases, respectively. The acetate fragment is extruded after precorrin-4 formation. This study combined with our recent structural studies on factor-IV (D. Thibaut, L. Debussche, D. Fréchet, F. Herman, M. Vuilhorgne, and F. Blanche, J. Chem. Soc. Chem. Commun. 1993:513-515, 1993) and precorrin-3B (L. Debussche, D. Thibaut, M. Danzer, F. Debu, D. Fréchet, F. Herman, F. Blanche, and M. Vuilhorgne, J. Chem. Soc. Chem. Commun. 1993:1100-1103, 1993) provides a first step-by-step picture of the sequence of the enzymatic reactions leading to the corrin ring in P. denitrificans.     

Resistenzphysiologische Grundlagen der evolutiven Kälteakklimatisation von Sproßpflanzen

Gradations in cold resistance of plants with different latitudinal and altitudinal distribution, and the various categories of chilling and freezing resistance are hypothesized to be evolutionary steps of adaptive responses to increasing low temperature stress and annual seasonality. The gradual lowering of the critical phase transition temperature of biomembrane lipids, the capacity of persistent supercooling of tissues, and the development of a dormancy linked freezing-tolerance are considered to be essential mechanisms resulting in improved acclimatation to low temperature climates.

     

Binding of adducts of NAD(P) and enolizable ketones to NAD(P)-dependent dehydrogenases

Carbonyl compounds such as alpha-ketoglutarate, pyruvate, oxaloacetate, butyraldehyde, acetaldehyde or acetone react with NAD or NADP to give adducts. Binding studies of adducts to dehydrogenases are performed by means of ultraviolet differential spectroscopy, circular dichroism and spectrofluorimetry. The dehydrogenases show a high degree of binding specificity toward the adducts which contain their specific oxidized substrate and their specific coenzyme. The high selectivity of the dehydrogenases for adducts is evidenced by binding studies of NAD(P)-pyruvate and NAD(P)-alpha-ketoglutarate adducts on glutamate dehydrogenase at pH 7.6 and 8.9. Evidence is presented showing that adducts bind to the active site of the enzymes.     

A bifunctional protein from Pseudomonas denitrificans carries cobinamide kinase and cobinamide phosphate guanylyltransferase activities.

The two consecutive activities of the cobalamin biosynthetic pathway that catalyze the conversion of cobinamide to cobinamide phosphate (cobinamide kinase) and of cobinamide phosphate to GDP-cobinamide (cobinamide phosphate guanylytransferase) were shown to be carried by the same protein in Pseudomonas denitrificans. This bifunctional protein was purified to homogeneity by high-performance liquid chromatography of extracts of a recombinant strain of this microorganism, and the sequence of the first 10 amino acid residues at the N terminus was determined. Both activities were specific to the coenzyme forms of the corrinoid substrates and exhibited an optimum pH at 8.8. Both ATP and GTP were shown to be in vitro gamma-phosphate donors for cobinamide kinase. However, competition experiments demonstrated that ATP was the preferred substrate, a result that can be explained in terms of the kinetic properties of the enzyme. Labeling experiments established that the phosphate group of cobinamide phosphate is quantitatively retained as the inner phosphate of GDP-cobinamide during the guanylyltransferase reaction. The native protein had an apparent molecular weight of 40,000, as estimated by gel filtration, and consisted of two identical subunits of Mr 20,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein had an isoelectric point of 5.35 and contained a high-affinity GTP-binding site (Kaff.(GTP) = 0.22 microM). Binding of GTP onto this site resulted in a marked increase of the affinity of cobinamide kinase for cobinamide. This property and other kinetic properties may regulate the enzyme and prevent the accumulation of cobinamide phosphate.     

The final step in the biosynthesis of hydrogenobyrinic acid is catalyzed by the cobH gene product with precorrin-8x as the substrate.

The final enzymatic reaction in the conversion of precorrin-6x to hydrogenobyrinic acid by cell-free protein preparations from Pseudomonas denitrificans was shown to be inhibited by hydrogenobyrinic acid. Use was made of this property to prepare the last biosynthetic precursor of hydrogenobyrinic acid, named precorrin-8x. Double-labeling experiments, mass spectrometry, and UV-visible light spectroscopy studies established that precorrin-8x was at the oxidation level of a corrin and differed from precorrin-6x by two additional methyl groups (presumably at C-5 and C-15) and decarboxylation of the acetic acid side chain at C-12. Precorrin-8x was not a corrin but had the same mass as hydrogenobyrinic acid, thus showing that this latter compound is synthesized from the former by a rearrangement. The enzyme catalyzing this rearrangement was purified 80-fold to homogeneity from a recombinant strain of P. denitrificans, sequenced at its N terminus, and shown to be encoded by the cobH gene. It was identical to the previously described hydrogenobyrinic acid-binding protein (F. Blanche, D. Thibaut, D. Frechet, M. Vuilhorgne, J. Crouzet, B. Cameron, G. Müller, K. Hlineny, U. Traub-Eberhard, and M. Zboron, Angew. Chem. Int. Ed. Engl. 29:884-886, 1990). This enzyme had a Km of 0.91 +/- 0.04 microM and a Vmax of 230 nmol h-1 mg-1 at pH 7.7 and was competitively inhibited by hydrogenobyrinic acid with a Ki of 0.17 +/- 0.01 microM. It is proposed that the cobH gene product is a mutase which transfers the methyl group from C-11 to C-12.     

Purification and characterization of cobyrinic acid a,c-diamide synthase from Pseudomonas denitrificans.

Cobyrinic acid a,c-diamide synthase, which catalyzes the conversion of cobyrinic acid to cobyrinic acid a,c-diamide via the intermediate formation of cobyrinic acid c-monoamide, was purified 155-fold to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans by high-performance liquid chromatography. The enzyme has an apparent molecular weight of 86,000 and consists of two identical subunits of Mr 45,000, as estimated by gel electrophoresis under denaturing conditions. Stepwise Edman degradation provided the N-terminal sequence of the first 15 amino acids. Glutamine was shown to be the preferred amino group donor (Km = 20.3 microM), but it could be replaced by ammonia (Km = 12 mM). The reaction was ATP dependent and exhibited a broad optimum pH around 7.3. Km values for (CN,aq)cobyrinic acid, (aq)2cobyrinic acid, and (CN,aq)cobyrinic acid c-monoamide were 160, greater than or equal to 250, and 71 microM, respectively. Hydrogenobyrinic acid and hydrogenobyrinic acid c-monoamide were shown to be much better substrates, with Km values of 0.41 and 0.21 microM, respectively.