Ryoko Iwamoto

Catalytic activities of [GADV]-peptides : Formation and establishment of [GADV]-protein world for the emergence of life

We have previously postulated a novel hypothesis for the origin of life, assuming that life on the earth originated from “[GADV]-protein world”, not from the “RNA world” (see Ikeharas review, 2002). The [GADV]-protein world is constituted from peptides and proteins with random sequences of four amino acids (glycine [G], alanine [A], aspartic acid [D] and valine [V]), which accumulated by pseudo-replication of the [GADV]-proteins. To obtain evidence for the hypothesis, we produced [GADV]-peptides by repeated heat-drying of the amino acids for 30 cycles ([GADV]-P30) and examined whether the peptides have some catalytic activities or not. From the results, it was found that the [GADV]-P30 can hydrolyze several kinds of chemical bonds in molecules, such as umbelliferyl-β-D-galactoside, glycine-p-nitroanilide and bovine serum albumin. This suggests that [GADV]-P30 could play an important role in the accumulation of [GADV]-proteins through pseudo-replication, leading to the emergence of life. We further show that [GADV]-octapaptides with random sequences, but containing no cyclic compounds as diketepiperazines, have catalytic activity, hydrolyzing peptide bonds in a natural protein, bovine serum albumin. The catalytic activity of the octapeptides was much higher than the [GADV]-P30 produced through repeated heat-drying treatments. These results also support the [GADV]-protein-world hypothesis of the origin of life (see Ikeharas review, 2002). Possible steps for the emergence of life on the primitive earth are presented.

The synthesis of 6-O-(2-acetamido-2-deoxy-β-D-glucopyranosyluronic acid)-D-glucose

Abstract Two routes for the synthesis of 6- O -(2-acetamido-2-deoxy-β- D -glucopyranosyluronic acid)- D -glucose ( 12 ) were studied. In the first, benzyl 6- O -(2-acetamido-2-deoxy-β- D -glucopyranosyl)-β- D -glucopyranoside ( 10 ) was obtained by condensation of benzyl 2,3,4-tri- O -acetyl-β- D -glucopyranoside (obtained via the 6- O -trityl derivative) with 3,4,6-tri- O -acetyl-2-deoxy-2-diphenylphosphorylamino-α- D -glucopyranosyl bromide, followed by removal of the diphenylphosphoryl and O -acetyl groups and subsequent N -acetylation. In the second route, 2-methyl-4,5-(3,4,6-tri- O -acetyl-2-deoxy-α- D -glucopyrano)-2-oxazoline was used as 2-acetamido-2-deoxy- D -glucose moiety in the condensation reaction to give benzyl 6- O -(2-acetamido-3,4,6-tri- O -acetyl-2-deoxy-β- D -glucopyranosyl)-2,3,4-tri- O -acetyl-β- D -glucopyranoside ( 11 ), and subsequent removal of O -acetyl groups gave compound 10 . Oxidation of the primary hydroxyl group of 10 gave benzyl 6- O -(2-acetamido-2-deoxy-β- D -glucopyranosyluronic acid)-β- D -glucopyranoside ( 8 ), which was converted by catalytic hydrogenolysis into the free disaccharide 12 .

Enzymatic synthesis of 2-keto-d-gluconate and 2-keto-d-galactonate from d-glucose and d-galactose with cell culture of Pseudomonas fluorescens and 2-keto-galactonate from d-galactono 1,4-lactone with partially purified 2-ketogalactonate reductase

Abstract 2-Keto- d -gluconate and 2-keto- d -galactonate were prepared from d -glucose (with a yield of 40%) and d -galactose (with a yield of 25%), respectively, with cell culture of Pseudomonas fluorescens . However, 2-keto- d -mannoate was not prepared in this method. The time courses of the reactions showed that 2-keto- d -gluconic acid and 2-keto- d -galactonic acid were produced from d -glucose and d -galactose through d -gluconate and d -galactonate, respectively. When using d -galactono 1,4-lactone as a starting material, 2-keto- d -galactonate was produced with partially purified NADP-dependent 2-ketogalactonate reductase from P. fluorescens . Some fundamental properties of the 2-ketogalactonate reductase were compared with those of 2-ketogluconate reductase from Acetobacter and Gluconobacter .

Purification and properties of D-glucosaminate dehydratase from Agrobacterium radiobacter

D-Glucosaminate dehydratase (EC 4.2.1.26)* catalyzes the conversion of D-glucosaminate (GlcNA) to 2-keto3deoxy-D-gluconate and ~monia;it requires pyridox~-S’-phosphate ~yridox~-P) as a cofactor and is analogous to serine dehydratase and threonine dehydratase. The enzyme was discovered independently by Merrick and Roseman [ 1 ] and by Imanaga [2] in the cells of unidentified bacteria in their studies of metabolism of D~luco~m~e. In the former case, the enzyme was formed inducibly by GlcNA, which was used as a sole source of carbon, and partially purified from the cell free extract [3]. It is the exclusive pyridoxal-P enzyme which acts specifically on an amino-sugar derivative. However, since the early works [l-3], little attention has been given to the enzyme. We here describe the purification of the GlcNAdehydrataae from the glucosamineadapted cells of Agrobacterium radiobacter to homogenity, and some of its properties.

Identification of an indispensable amino acid for ppGpp synthesis of Escherichia coli SpoT protein.

Amino acid substitutions were introduced into a structurally flexible and highly conserved region of Escherichia coli SpoT protein. SpoT protein changed from Asp to Ala at the 293rd position did not restore cell growth of E. coli CF8295 (Δ relA, Δ spoT) and did not accumulate ppGpp in the cell, suggesting that the Asp293 is indispensable for ppGpp synthesis of the protein.

Purification and Characterization of d-Glucosaminate Dehydratase from Pseudomonas fluorescens

d-Glucosaminate dehydratase (EC 4.2.1.26) from Pseudomonas fluorescens (IFO 14808) was purified to homogeneity, as judged by the criterion of a single band on disc-gel electrophoresis. The enzyme (molecular weight 61,000) consisted of two subunits identical in molecular weight (about 30,000). Pyridoxal 5′-phosphate was an essential cofactor for the enzyme.The enzyme catalyzed the dehydration of d-glucosaminate and of several d- and l-hydroxyamino acids. Especially when the concentration of substrate was low, d-serine was dehydrated at a rate similar to the rate of dehydration of d-glucosaminate. If both substrates were added to the reaction mixture at the same time, the rate of the reaction was additive until their individual concentrations reached to 0.2 mM level. However, as the concentrations of both the substrates were increased, the rate fell below the rate recorded with d-glucosaminate only. The kinetics of this reaction (in the presence of 1~10mm d-GIcNA) demonstrate that d-glucosaminate dehydratas...

Stereochemistry of an α,β-elimination reaction by D-glucosaminate dehydratase

The proton NMR analysis of D‐glucosaminate dehydratase reaction in D2O revealed the incorporation of a deuterium atom at C‐3 carbon of the product, 2‐keto‐3‐deoxy‐D‐gluconate. Based on the chemical shift of C‐3 proton of the product and the coupling constant characteristic for the C‐3 and C‐4 axial‐axial coupling in the 2C5 pyranose conformation, the deuterium is in the pro‐S position. Thus, the dehydration of D‐glucosaminate by the enzyme proceeds in a retention mode at C‐3 carbon. Kinetic parameters show that the rate‐determining step is the abstraction of α‐proton from the substrate.