Mamoru Honma

Insight into a natural Diels-Alder reaction from the structure of macrophomate synthase.

The Diels–Alder reaction, which forms a six-membered ring from an alkene (dienophile) and a 1,3-diene, is synthetically very useful for construction of cyclic products with high regio- and stereoselectivity under mild conditions. It has been applied to the synthesis of complex pharmaceutical and biologically active compounds. Although evidence on natural Diels–Alderases has been accumulated in the biosynthesis of secondary metabolites, there has been no report on the structural details of the natural Diels–Alderases. The function and catalytic mechanism of the natural Diels–Alderase are of great interest owing to the diversity of molecular skeletons in natural Diels–Alder adducts. Here we present the 1.70 Å resolution crystal structure of the natural Diels–Alderase, fungal macrophomate synthase (MPS), in complex with pyruvate. The active site of the enzyme is large and hydrophobic, contributing amino acid residues that can hydrogen-bond to the substrate 2-pyrone. These data provide information on the catalytic mechanism of MPS, and suggest that the reaction proceeds via a large-scale structural reorganization of the product.

Diverse expression profiles of 21 rice peroxidase genes

Secretory class III plant peroxidases (POXs) catalyze the oxidation of various reductants, and are encoded by a large multigene family. In rice, 42 independent expressed sequence tags for POXs have been identified. By RNA gel blot analysis using specific probes, we show here that 21 rice POX genes are unique in their developmental, organ specific and external stimuli‐responsive expression. This would suggest that encoded POX isoenzymes are involved in a broad range of physiological processes in rice plants, individually.

Purification and properties of acid phosphatase secreted from lupin roots under phosphorus-deficiency conditions

Abstract When lupin plants were grown in phosphorus-deficient nutrient solutions, the activity of acid phosphatase secreted from their roots remarkably increased in comparison with that under adequate phosphorus conditions. Based on activity staining of acid phosphatase on IEF gels, many isozymes were present in roots and leaves, but one of these was mainly secreted into the rhizosphere. This main acid phosphatase secreted from roots was purified from the phosphorus-deficient nutrient solution where lupin was grown, using ion exchange chromatography with DEAE-Sepharose CL-6B, gel filtration with Bio-Gel P-200, and preparative-PAGE. The molecular weight of the purified enzyme was estimated to be 72,000 by SDS-PAGE and approximately 140,000 by gel filtration. Thus, this enzyme was considered to be a homo-dimer. The isoelectric point and the optimum pH of the enzyme were 4.7 and 4.3, respectively.

An HR-Induced Tobacco Peroxidase Gene Is Responsive to Spermine, but Not to Salicylate, Methyl Jasmonate, and Ethephon

In Tobacco mosaic virus (TMV)-infected tobacco plants carrying the N resistance gene, a hypersensitive reaction or response (HR) occurs to enclose the virus in the infected tissue. Although a contribution of peroxidases to the resistance has been proposed, no evidence has been presented that tobacco peroxidase genes respond to HR. Here, we describe the HR-induced expression of a tobacco peroxidase gene (tpoxC1) whose induction kinetics were slightly different from those of acidic and basic tobacco pathogenesis-related (PR) protein genes. Interestingly, tpoxC1 was insensitive to the inducers of PR genes such as salicylic acid, methyl jasmonate, and ethephon. Spermine activated tpoxC1 gene expression at a low level and both acidic and basic PR gene expression at a considerably higher level. These results indicate that the induced expression of tpoxC1 is regulated differently from that of classical tobacco PR genes in the N gene-mediated self-defense system in tobacco plants.

ENZYMATIC ACTIVITY AND PARTIAL PURIFICATION OF SOLANAPYRONE SYNTHASE : FIRST ENZYME CATALYZING DIELS-ALDER REACTION

In cell-free extracts of Alternaria solani, an enzymatic activity converting prosolanapyrone II to solanapyrones A and D via oxidation and subsequent Diels-Alder reaction has been found. Chromatography with DEAE-Sepharose provided two active fractions, pools 1 and 2. The former fraction converted prosolanapyrone II to solanapyrones A and D in a ratio of 2.2:1 with optical purities of 99% and 45% ee, respectively. The latter fraction did so in a ratio of 7.6:1 with 99% and nearly 0% ee, respectively. The enzyme partially purified from pool 2 native molecular weight of 40-62 kD and a pl of 4.25. The high reactivity of prosolanapyrone III in aqueous solution and the chromatographic behavior of the enzyme in pool 2 suggest that a single enzyme catalyzes both the oxidation and Diels-Alder reaction.

Xylem-specific expression of wound-inducible rice peroxidase genes in transgenic plants.

A peroxidase gene, poxA, was isolated from a rice (Oryza sativa L.) genomic library. The gene consists of four exons whose combined sequences were identical to that of the prxRPA mRNA whose levels were dramatically stimulated by wounding as well as by treatment of rice shoots with ethephon or UV irradiation [H. Ito, F. Kimizuka, A. Ohbayashi, H. Matsui, M. Honma, A. Shinmyo, Y. Ohashi, A.B. Caplan, R.L. Rodriguez, Molecular cloning and characterization of two complementary DNAs encoding putative peroxidases from rice (Oryza sativa L.) shoots, Plant Cell Rep. 13 (1994) 361-366]. The temporal and spatial expression properties of the poxA gene promoter as well as that from a second related peroxidase gene, poxN, were analyzed in transgenic tobacco and rice plants using the uidA gene as a reporter. In transgenic tobacco, UV- and wound-responsive cis-elements were located within 144 bp from the translational start codon of the poxA gene. The poxN promoter, however, was inactive in the heterologous host as no significant GUS activity was evident. On the other hand, chimeric uidA genes containing 2.2 kb of the poxA promoter or 1.4 kb of poxN promoter were active in transgenic rice plants. Both peroxidase promoters directed GUS activities in a spatial and tissue specific manner coincident with the expression patterns exhibited by their mRNAs. Histochemical analysis of transgenic rice plants showed that both peroxidase genes are expressed in the vascular bundles of the shoot apex and lamina joint, and in xylem-parenchyma cells of the leaf blade and sheath.

Purification and characterization of phytase induced in tomato roots under phosphorus-deficient conditions

Abstract Phytase (myo-inositol hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from roots of tomato plants grown under phosphorus-deficient conditions using five purification schemes. The phytase was successfully separated from the major acid phosphatase to an electrophoretic homogeneity. The native molecular weight of this enzyme was estimated to be about 164 kD by Bio-Gel P-200 gel filtration. The molecular weight of the subunit on SDS-PAGE was approximately 82 kD, indicating that the native form of the enzyme was a homodimer. The isoelectric point of tomato phytase was about 5.5. The enzyme exhibited a high affinity for phytic acid (K m = 38 μM), and was strongly inhibited by phosphate, molybdate and fluoride. Among other characteristics of tomato phytase, the pH and temperature optima were 4.3 and 45°C, respectively. Tomato phytase contained a fairly high concentration of aspartic, glutamic acid and glycine residues.

Two starch-branching-enzyme isoforms occur in different fractions of developing seeds of kidney bean

The nature and enzymic properties of starch-branching enzyme (SBE) are two of the dominant factors influencing the fine structure of starch. To understand the role of this enzymes activity in the formation of starch in kidney bean (Phaseolus vulgaris L.), a study was undertaken to identify the major SBE sequences expressed during seed development and to characterize the enzymic properties of the coded recombinant enzymes. Two SBE cDNA species (designated pvsbe2 and pvsbe1) that displayed significant similarity (more than 70%) to other family A and B SBEs respectively were isolated. Northern blot analysis revealed that pvsbe1 and pvsbe2 were differentially expressed during seed development. pvsbe2 showed maximum steady-state transcript levels at the mid-stage of seed maturation, whereas pvsbe1 reached peak levels at a later stage. Western blot analysis with antisera raised against both recombinant proteins (rPvSBE1 and rPvSBE2) showed that these two SBEs were located in different amyloplast fractions of developing seeds of kidney bean. PvSBE2 was present in the soluble fraction, whereas PvSBE1 was associated with the starch granule fraction. The differences in location suggest that these two SBE isoenzymes have different roles in amylopectin synthesis in kidney bean seeds. rPvSBE1 and rPvSBE2 were purified from Escherichia coli and their kinetic properties were determined. The affinity of rPvSBE2 for amylose (K(m) 1.27 mg/ml) was lower than that of rPvSBE1 (0.46 mg/ml). The activity of rPvSBE2 was stimulated more than 3-fold in the presence of 0.3 M citrate, whereas rPvSBE1 activity was not affected. The implications of the enzymic properties and the distribution of SBEs and amylopectin structure are discussed.

1-Aminocyclopropane-1-carboxylate (ACC) Deaminase Induced by ACC Synthesized and Accumulated in Penicillium citrinum Intracellular Spaces

We have already described how 1-aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of the plant hormone ethylene, is synthesized in Penicillium citrinum through the same reaction by the catalysis of ACC synthase [EC 4.4.1.14] as in higher plants. In addition, ACC deaminase [EC 4.1.99.4], which degrades ACC to 2-oxobutyrate and ammonia, was also purified from this strain. To study control of induction of ACC deaminase in this organism, we have isolated and analyzed the cDNA of P. citrinum ACC deaminase and studied the expression of ACC deaminase mRNA in P. citrinum cells. By the analysis of peptides from the digests of the purified and modified ACC deaminase with lysylendopeptidase, 70% of its amino acid sequences were obtained. These amino acid sequences were used to identify a cDNA, consisting of 1,233 bp with an open reading frame of 1,080 bp encoding ACC deaminase with 360 amino acids. The deduced amino acids from the cDNA are identical by 52% and 45% to those of enzymes of Pseudomonas sp. ACP and Hansenula saturnus. Through Northern blot analysis, we found that the mRNA of ACC deaminase was expressed in P. citrinum cells grown in a medium containing 0.05% L-methionine. These findings suggest that ACC synthesized by ACC synthase and accumulated in P. citrinum intracellular spaces can induce the ACC deaminase that degrades the ACC.

Reaction intermediate structures of 1-aminocyclopropane-1-carboxylate deaminase: insight into PLP-dependent cyclopropane ring-opening reaction

The pyridoxal 5′-phosphate-dependent enzymes have been evolved to catalyze diverse substrates and to cause the reaction to vary. 1-Aminocyclopropane-1-carboxylate deaminase catalyzes the cyclopropane ring-opening reaction followed by deamination specifically. Since it was discovered in 1978, the enzyme has been widely investigated from the mechanistic and physiological viewpoints because the substrate is a precursor of the plant hormone ethylene and the enzymatic reaction includes a cyclopropane ring-opening. We have previously reported the crystal structure of the native enzyme. Here we report the crystal structures of the two reaction intermediates created by the mutagenesis complexed with the substrate. The substrate was validated in the active site of two forms: 1) covalent-bonded external aldimine with the coenzyme in the K51T form and 2) the non-covalent interaction around the coenzyme in the Y295F form. The orientations of the substrate in both structures were quite different form each other. In concert with other site-specific mutation experiments, this experiment revealed the ingenious and unique strategies that are used to achieve the specific activity. The substrate incorporated into the active site is reactivated by a two-phenol charge relay system to lead to the formation of a Schiff base with the coenzyme. The catalytic Lys51 residue may play a novel role to abstract the methylene proton from the substrate in cooperation with other factors, the carboxylate group of the substrate and the electron-adjusting apparatuses of the coenzyme.