Gennady P. Moiseyev

High sequence similarity between a ribonuclease from ginseng calluses and fungus-elicited proteins from parsley indicates that intracellular pathogenesis-related proteins are ribonucleases

A ribonuclease from a callus cell culture of Panax ginseng C.A. Mey strain R1 was isolated. A pure protein with an apparent molecular mass of 18 kDa was obtained. The N-terminal sequences of the protein and of the C-terminal CNBr peptide were determined. No homology with other ribonucleases was found, but there was 60–70% sequence identity with two intracellular pathogenesis-related (IPR) proteins from parsley, indicating that not only these two proteins, but also homologous IPR proteins identified in other plant species are ribonucleases.

Primary structures of two ribonucleases from ginseng calluses - New members of the PR-10 family of intracellular pathogenesis-related plant proteins

The amino acid sequences of two ribonucleases from a callus cell culture of Panax ginseng were determined. The two sequences differ at 26% of the amino acid positions. Homology was found with a large family of intracellular pathogenesis‐related proteins, food allergens and tree pollen allergens from both dicotyledonous and monocotyledonous plant species. There is about 30% sequence difference with proteins from species belonging to the same plant order (Apiales: parsley and celery), 60% with those from four other dicotyledonous plant orders and about 70% from that of the monocotyledonous asparagus. More thorough evolutionary analyses of sequences lead to the conclusion that the general biological function of members of this protein family may be closely related to the ability to cleave intracellular RNA and that they have an important role in cell metabolism. As the three‐dimensional structure of one of the members of this protein family has been determined recently [Gajhede et al., Nature Struct Biol 3 (1996) 1040–1045], it may be possible to assign active‐site residues in the enzyme molecule and make hypotheses about its mode of action. Structural features in addition to the cellular site of biosynthesis indicate that this family of ribonucleases is very different from previously investigated ones.

MUTATIONAL ANALYSIS OF THE ACTIVE SITE OF RNASE OF BACILLUS INTERMEDIUS (BINASE)

To elucidate the functional role of some residues in the active site of binase, the extracellular ribonuclease of Bacillus intermedius, we used site‐directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant His101Glu is 2.0–2.7% of that for native enzyme. The decrease in activity is determined mainly by the decrease in molecular rate constant k cat with almost unchanged affinity of the enzyme for the substrate, characterized by K M. This is the expected result if His101 acts as an general acid, donating a proton to the leaving group on cleavage of a phosphodiester bond. The replacement of Lys26 by Ala causes a reduction in the enzyme activity to 13—33%, depending on the substrate. The activity decreases are due to changes in both k cat and K M for poly(I) and poly(A) but in k cat alone for GpA. In the latter case the effect is far less than that seen in the homologous mutation in the closely related enzyme, barnase.

Dissociation constants and thermal stability of complexes of Bacillus intermedius RNase and the protein inhibitor of Bacillus amyloliquefaciens RNase

Binase, the extracellular ribonuclease of Bacillus intermedius, is inhibited by barstar, the natural protein inhibitor of the homologous RNase, barnase, of B. intermedius. The dissociation constants of the binase complexes with barstar and its double Cys40.82 Ala mutant are about 10−12 M, only 5 to 43 times higher than those of the barnase‐barstar complex. As with barnase, the denaturation temperature of binase is raised dramatically in the complex. Calorimetric studies of the formation and stability of the binase‐barstar complex show that the binase reaction with barstar is qualitatively similar to that of barnase but some significant quantitative differences are reported.

Determination of the nucleotide conformation in the productive enzyme-substrate complexes of RNA-depolymerases

© 1997 Federation of European Biochemical Societies.

Primary structure and catalytic properties of extracellular ribonuclease of bacillus circulans

A complete amino acid sequence of extracellular Bacillus circulans RNase was established and compared with a structure of B. amyloliquefaciens RNase. Gln15, Gly65 and Gln104 in B. amyloliquefaciens RNase were found to be replaced by Leu, Ala and Lys, respectively, in B circulans RNase. Catalytic properties of B. circulans RNase were studied.

Specificity of guanylic RNases to polynucleotide substrates

Kinetic parameters kcat and KM were measured for cleavage of poly I, poly A, poly U, poly C and poly I poly C by guanyl-specific RNases Sa, Pb1 and T1 and compared with that of guanyl-preferential RNase Bi. Catalytic efficiencies of the investigated enzymes to polynucleotide substrates vary considerably. The structural basis for specificity of these RNases is discussed. A hypothesis is suggested that Ser-56 plays an important role in recognition of poly A by RNase Bi.

A novel guanyl‐preferable ribonuclease of Bacillus polymyxa: Isolation and characterization of the enzyme

Three forms of the extracellular alkaline ribonuclease of B. polymyxa (RNases Bpo I, Bpo II and Bpo III) were obtained in the homogeneous states. Relative molecular weights, N‐ and C‐terminal amino acid sequences of the proteins were determined. The two higher‐molecular weight forms of the enzyme (RNases Bpo I and Bpo III) were shown to be precursor molecules which differed from the mature B. polymyxa RNase (RNase Bpo II) by the presence of a extra twelve and six amino acid residues at the amino terminus, respectively. The study of catalytic properties of the enzyme elucidated that the extent of specificity of B. polymyxa RNase in respect to high‐polymeric substrates is different from that of other natural Bacillus RNases. Amino acid composition of B. polymyxa RNase was established and structural similarity within family of Bacillus RNases is discussed. B. polymyxa RNase was shown to be inhibited by intracellular protein inhibitor of B. amyloliquefaciens RNase with the dissociation constant of 2.7×10‐12 M.

Correlative variations of the free energies for enzyme-substrate complex formation and the transition-state stabilization for RNases

It was found for RNases of different specificities that changes in the free energy for substrate‐enzyme binding induced by variations in the nucleotide base structure are accompanied by proportional changes in k cat/K m. This was considered to be a consequence of the strain in the enzyme‐substrate complex.