Kazuko Ohgi

Crystal and molecular structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus

The crystal structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.5 Å resolution by the multiple isomorphous replacement method. The crystal structure was refined by simulated annealing with molecular dynamics. The current crystallographic R‐factor is 0.200 in the 10—2.5 Å resolution range. The molecular structure which is completely different from the known structures of RNase A and RNase T1 consists of six α‐helices and seven β‐strands, belonging to the α+β type structure. Two histidine and one glutamic acid residues which were predicted as the most probably functional residues by chemical modification studies are found to be clustered. The steric nature of the active site taken together with the relevant site‐directed mutagenesis experiments (Irie et al.) indicates that: (i) the two histidine residues are the general acid and base; and (ii) an aspartic acid residue plays a role of recognizing adenine moiety of the substrate.

Heat stable ssDNA/RNA-binding activity of a wheat cold shock domain protein

The cold‐induced wheat WCSP1 protein belongs to the cold shock domain (CSD) protein family. In prokaryotes and eukaryotes, the CSD functions as a nucleic acid‐binding domain. Here, we demonstrated that purified recombinant WCSP1 is boiling soluble and binds ss/dsDNA and mRNA. Furthermore, boiled‐WCSP1 retained its characteristic nucleic acid‐binding activity. A WCSP1 deletion mutant, containing only a CSD, lost ssDNA/RNA‐binding activity; while a mutant containing the CSD and the first glycine‐rich region (GR) displayed the activity. These data indicated that the first GR of WCSP1 is necessary for the binding activity but is not for the heat stability of the protein.

A New Type of RNase T2 Ribonuclease in Two Basidiomycetes Fungi, Lentinus edodes and Irpex lacteus

Two new RNase T2 Ribonucleases, RNase Le37 and Irp3, with a molecular mass of 45 kDa, have been isolated from Basidiomycetes fungi, Lentinus edodes and Irpex lacteus, respectively. The ribonucleases consisted of three domains: an RNase active domain, a Ser/Thr rich domain similar to that of many fungal glycanhydrolases, and a C-terminal 10 kDa domain similar to that of RNase Rny1 in yeast. The locations of hydrophobic amino acids and Pro in the 10 kDa domain of the two basidiomycetous enzymes are very similar to those of RNase Rny1, indicating that these domains may have similar roles.

Amino Acid Sequence of an Unique Ribonuclease with a C-Terminus rich in O-Glycosylated Serine and Threonine from Culture Medium of Lentinus edodes

The mushroom Lentinus edodes produces three base-non-specific and acid ribonucleases, RNases Le2, Le37, and Le45. The latter two are excreted from mycelia into the medium. The primary structure of RNase Le37, which had a molecular mass of 37 kDa, was sequenced. It was a member of the RNase T2 family, as is RNase Le2. RNase Le37 was some 30 amino acid residues longer at the C-terminal end than RNase Le2. The C-terminal region of RNase Le37 was rich in O-glycosylated serine and threonine. In fungal glucoamylases and chitinases, which hydrolyze raw-starch and chitin, respectively, have structures resembling the structure of the C-terminal of RNase Le37.

Enzymatic Properties of Phenylalanine101 Mutant Enzyme of Ribonuclease Rh from Rhizopus niveus

To investigate the role of Phe101, a component of a base recognition site (B2 site) of a base-nonspecific RNase Rh from Rhizopus niveus, we prepared several enzymes mutated at this position, F101W, F101L, F101I, F101A, F101Q, F101R, and F101K, and their enzymatic activities towards RNA, 16 dinucleoside phosphates, and 2′, 3′-cyclic pyrimidine nucleotides were measured. Enzymatic activity toward RNA of F101W, F101L, and F101I were about 7, 20, and 3.8% of the native enzyme, respectively, and those of the other mutants were less than 1% of the RNase Rh. Similar results were also obtained with GpG as substrate. Thus, it was concluded that Phe101 is a very important residue as a component of the B2 site of RNase Rh, and its role could be replaced by Leu, then Trp and Ile, though in less effectively. The results suggested that some kind of interaction between B2 base and the side chain of amino acid residue at the 101th position, such as π/π or CH/π interaction is very important for the enzymatic activity of RNase Rh. The mutation of Phe101 markedly affected the enzymatic activity toward dinucleoside phosphates and polymer substrates, but only moderately the rate of hydrolysis of cyclic nucleotides, indicating the presence of secondary effect of the mutation on B1 site.

Crystallization of a new class of microbial ribonuclease from Rhizopus niveus

Crystals of ribonuclease Rh, a new class of microbial ribonuclease from Rhizopus niveus, were obtained from polyethylene glycol 8000 solution by a vapour diffusion technique in the hanging drop mode. Two crystal forms, type I and type II, were obtained from the same droplet solution. Both forms belong to the space group P2(1)2(1)2(1), but their cell dimensions are markedly different: a = 68.3 A, b = 73.0 A, c = 50.0 A for type I and a = 67.5 A, b = 72.3 A, c = 44.2 A for type II. The type I crystals diffract beyond 2.0 A resolution and are suitable for X-ray structure analysis at high resolution.

On a Salmon (Onchorhynchus keta) Liver RNase, Belonging to RNase T2 Family: Primary Structure and Some Properties

A base-nonspecific and acid ribonuclease (RNase Ok2) was purified from the liver of a salmon (Oncorhnchus keta) to a homogeneous state by SDS–PAGE. The primary structure of RNase Ok2 was determined by protein chemistry and molecular cloning. The RNase Ok2 was a glycoprotein and consisted of 216 amino acid residues. Its molecular mass of protein moiety was 25,198, and its amino acid sequence showed that it belongs to the RNase T2 family of enzymes. The optimal pH of RNase Ok2 was around 5.5. The base preferences at the B1 and B2 sites were estimated from the rates of hydrolysis of 16 dinucleoside phosphates to be G>>A>U, C, and G>A>U>C respectively. In this enzyme, one of the three histidine residues which have been thought to be important for catalysis of RNase Rh, a typical RNase of this family of enzymes, His104 was replaced by tyrosine residue. Based on the results, the role of H104, which has been proposed to be a phosphate binding site with a substrate, was reconsidered, and we proposed a revised role of this His residue in the hydrolysis mechanism of RNase T2 family enzymes.

Enzymatic Properties of Glutamine 32 Mutants of RNase Rh from Rhizopus niveus, a Trial to Alter the Most Preferential Inter-nucleotidic Linkages of RNase Rh

In order to investigate the effects of mutation of Gln32, a component of a base recognition site (B2 site) of a base-nonspecific RNase from Rhizopus niveus, we prepared several enzymes mutant at this position, Q32F, Q32L, Q32V, Q32T, Q32D, Q32N, and Q32E, and their enymatic activities toward RNA and 16 dinucleoside phosphates were measured. Enzymatic activities of the mutant enzymes towards RNA were between 10-125% of the native enzyme. From the rates of hydrolysis of 16 dinucleoside phosphates by mutant enzymes, we estimated the base specificity of both B1 and B2 sites. The results indicated that mutation of Gln32 to Asp, Asn, and Glu caused the B2 site to prefer cytosine more and to a less extent, to prefer uracil (Q32N), and that Q32F made the enzyme more guanine-base preferential. The results suggested that we are able to construct an enzyme that preferentially cleaves internucleotidic linkages, at the 5′-side of cytosine residues (Q32D, Q32N, and Q32E) and guanine residues (Q32F and Q32T), thus, cleaves purine-C(Q32D, Q32N, Q32E) and GpG and ApG (Q32F, and Q32T) most easily. The results seemed to suggest converting a base-non-specific RNase to a base-specific one.

Amino Acid Sequence of a Nuclease (Nuclease Le1) from Lentinus edodes

The fruit bodies of Lentinus edodes produce two acid nucleases, nucleases Le1 and Le3, both of which are thought to be candidates for the enzymes producing a tasty substance, 5′-GMP. To obtain the basic information on the mechanism of production of 5′-GMP, and structure-function relationship of these nucleases, the primary structure of nuclease Le1 was estimated by both protein chemistry and gene cloning. Nuclease Le1 is a glycoprotein and consists of 290 amino acid residues, and about 2 and 6 residues of hexosamine and neutral sugar, respectively. The nucleotide sequence of cDNA and genomic DNA encoding nuclease Le1 indicated the presence of 20 amino acid residues of a signal peptide. Nuclease Le1 has 115 and 108 residues of identical amino acid residues with nucleases P1 and S, respectively. The amino acid residues concerning the coordination with Zn2+ in nuclease P1 are all conserved in nuclease Le1. Nuclease Le1 contains 8 half-cystine residues and 4 of them are located at the same places as those of nucleases P1 and S.

The structure of the asparagine-linked sugar chains of bovine brain ribonuclease

The asparagine-linked sugar chains of bovine brain ribonuclease were quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. After N-acetylation, they were converted into radioactively-labeled oligosaccharides by NaB3H4 reduction. The radioactive oligosaccharide mixture was fractionated by ion-exchange chromatography, and the acidic oligosaccharides were converted into neutral oligosaccharides by sialidase digestion. The neutral oligosaccharides were then fractionated by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exoglycosidase digestion in combination with methylation analysis revealed that bovine brain ribonuclease showed extensive heterogeneity. It contains bi- and tri-antennary, complex-type oligosaccharides having alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-beta-D- GlcpNAc-(1----4)-[alpha-L-Fucp-(1----6)]-D-GlcNAc as their common core. Four different outside oligosaccharide chains, i.e., beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----, alpha-Neu5Ac-(2----6)-beta-D- Galp-(1----4)-beta-D-GlcpNAc-(1----, alpha-Neu5Ac-(2----3)-beta-D-Galp-(1----4)- beta-D-GlcpNAc-(1----, and alpha-D-Galp-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----, were found. The preferential distribution of the alpha-D-Galp-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc group on the alpha-D-Manp-(1----6) arm is a characteristic feature of the sugar chains of this enzyme.