Susumu Hiragai' 4, Hireyuki Ito i, Katsutomo Sasaki i' 4, Hiromoto Yamakawa2' 4, Ichiro Mitsuhara3' 4, Hiroaki Toshima i, Hirokazu Matsui i, Mamoru Honma i and Yuko 0hashi3i 4i S i Department ofApptiedBioscience, Graduate Schoot ofAgriculture, Hbkkaido University, Stmporo, 060-8589 Jopan 2 institute ofBiotogical Sciences, Uhiversity of 7lsukuba, Tlsukuba, Ibaraki, 3C15-85Z2 Japan i DE:partment ofMbiecular Geneties, Ntitional Ihstitute ofAgrobiological Resources, 7lsukuba, Ibaraki, 305-86{12 Jlu)an ` Core Research for Evolutional Science and TZichnology rCREST), Chiyoda-ku, fokyo, IOJ-O062 Japan

mation to modify an existing enzyme for adcling another function or to create a new enzyme that can transform molecules in a stereoeontrolled manner, Yamamoto's group4'5} have previously shown that macrophomic acid (1)6] and the phytotoxin, pyrenochaetic acid (2),7) were biotransformed from the corresponding 2-pyrones, 3 and 4 (pyrenocine A8,9)), and a C3-acid precursor by the fungus, Macrophoma commelinae, as shown in Scheme 1, We have recently determined the C3-precursor to be an oxalacetate, and have purified and characterized the enzyme named as macrophomate synthase,ie"i2) This single enzyme catalyzes multiple reactions of oxalacetate with 2-pyrone 3 to afford benzoate 1 via the formation of two C-C bonds concomitant with two decarboxylation and dehydration reactions. The addition of the C3-acid to polyketides,i]} terpenoidsi4,T5) and fatty acid derivatives'6] is often to loe found in the biosynthesis of a secondary metabolite, There are two modes of condensation pattern, C-C bond formation at C-2i`) of the C3-acid and formation at C-3.i3'i5} In some cases, an oxalacetate has been proposed to be the Crunit precursor.i4,T6) Although there are several proposals for the formation of such metabolites, none of them has been established at an enzyme level, In the case of the conversion catalyzed by macrophomate synthase, condensation oceurs at both the C-2 and C-3 positions, and this type of aromatic ring formation is totally different from the common routes via the shikimate and polyketide pathways. Previous studies on biotransformation with the whole cell of M. commetinae have shown that various 2-pyrones were converted into the corresponding benzoates,j) To test the ability of macrophomate synthase to accept various pyrones as

Macrophomate synthase (MPS) is an enzyme that catalyzes an extraordinarily complex conversion reaction, including two decarboxylations, two carbon-carbon bond formations and a dehydration, to form the benzoate analogue macrophomate from a 2-pyrone derivative and oxalacetate. Of these reactions, the two carbon-carbon bond formations are especially noteworthy because previous experiments have indicated that they proceed via a Diels-Alder reaction, one of the most widely used reactions in organic synthesis. The structural evidence that MPS catalyzes an intermolecular Diels-Alder reaction has been reported recently [Ose et al. (2003), Nature (London), 422, 185-189]. Interestingly, the tertiary structure as well as the quaternary structure of MPS are similar to those of 2-dehydro-3-deoxygalactarate (DDG) aldolase, a carbon-carbon bond-forming enzyme that catalyzes the reversible reaction of aldol condensation/cleavage. Here, the structure of MPS is described in detail and compared with that of DDG aldolase. Both enzymes have a (beta/alpha)(8)-barrel fold and are classified as belonging to the enolase superfamily based on their reaction strategy. The basic principles for carbon-carbon bond formation used by both MPS and DDG aldolase are the same with regard to trapping the enolate substrate and inducing subsequent reaction. The major differences in the active sites between these two enzymes are the recognition mechanisms of the second substrates, 2-pyrone and DDG, respectively.

1-Aminocyclopropane-1-carboxylate deaminase (ACCD) is a pyridoxal 5′-phosphate dependent enzyme that shows deaminase activity toward ACC, a precursor of plant hormone ethylene. ACCD from some soil bacteria has been reported to be able to break the cyclopropane ring of ACC to yield α-ketobutyrate and ammonia. We reported the crystal structure of ACCD from the yeast Hansenula saturnus in the absence/presence of substrate ACC, and proposed its ingenious reaction mechanisms. In order to study the enzyme further, we overexpressed the ACCD homologue protein (phAHP) from the fully decoded hyperthermophilic archearon, Pyrococcus horikoshii OT3. However, phAHP does not show ACCD activity at high temperature as well as at room temperature, though it has significant sequence similarity. Instead of ACCD activity, the GC–MS analysis and enzymatic method show that phAHP has deaminase activity toward l and d-serine. Here, we present the crystal structures of the native and ACC-complexed phAHP. The overall topology of the phAHP structure is very similar to that of ACCD; however, critical differences were observed around the active site. Here, the differences of enzymatic activity between phAHP and ACCD are discussed based on the structural differences of these two proteins. We suggest that the catalytic disagreement between these two enzymes comes from the difference of the residues near the pyridine ring of pyridoxal 5′-phosphate (PLP), not the difference of the catalytic residues themselves. We also propose a condition necessary in the primary sequence to have ACCD activity.

Starches from kidney bean (Phaseolus vulgaris L. cv. Toramame) seeds at the immature, premature, mature stages of development were examined. The starch content increased from 94, 219 to 265 mg per seed. Starches showed the C(a)-crystalline type composed of small (<5 micrometer) and large (10-35 micrometer) granules, with the large granules largely increasing with maturity. The amylose content increased from 21, 26 to 27%, and rapid viscograms and DSC thermograms suggested that the mature-stage starch was gelatinized with ease. The amylose increased in size from DPn 820, 1000 to 1080 and a number of chains per molecule (NC) from 3.3, 4.2 to 4.5. The branched amylose was a minor component (11-18% by mole) with NC 20-22. The amylopectin was similar in CL (23), beta-amylolysis limit (59%), and chain-length distribution, but reduced in size (DPn 17,100-5270) and increased in content of phosphorus (114-174 ppm) with an increase in the amount of phosphorus linked to C-6 of the glucose residue (8-66%).

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 conditions1. It has been applied to the synthesis of complex pharmaceutical and biologically active compounds2. Although evidence3,4,5,6,7 on natural Diels–Alderases has been accumulated in the biosynthesis of secondary metabolites8, 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 adducts8. Here we present the 1.70 Å resolution crystal structure of the natural Diels–Alderase, fungal macrophomate synthase (MPS)3, 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.

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.

Pectate lyases are the major pectinases that play a key role in the development of the soft-rot disease.Besides in phytopathogens, pectin depolymerization has also been reported in non-pathogenic plant associated bacteria such as the N2fixing endosymbiont Rhizobium and the N2-fixing soil bacterium Azospirillum irakense.A gene from A. irakense encoding a pectate lyase (termed PelA) was isolated by heterologous expression of the gene in Escherichia coli.Analysis of the corresponding amino acid sequence revealed no homology to other bacterial, plant and fungal pectinases leading to the classification of the enzyme in a new pectate lyase family (family 10).The A. irakense PelA has been crystallized using the hanging-drop vapor diffusion method at 277K.These crystals are hexagonal with cell dimensions of a = b = 85.55 Å, c = 230.13Å, γ = 120°, and space group P6522 having one molecule per asymmetric unit2.Diffraction data to a resolution of 1.97 Å were collected at synchrotron facilities, as well as a three-wavelengths MAD data set on a Hg derivate crystal to a resolution of 2.6 Å.The preliminary structural results show that PelA does not have the characteristic parallel β-helix fold of the polysaccharide pectate lyase families.References

A putative α-glucosidase gene was isolated from the genomic library of Brevibacterium fuscumvar. dextranlyticum strain 0407. The gene, designated dexG, was located upstream of isomaltotriodextranase gene (dexT). The dexG contained an open reading frame of 1725 bp, and its deduced amino acid sequence (DexG) showed a high homology with the enzymes belonging to α-glucosidase Family I and I-like, especially oligo-l, 6-glucosidase from Bacillus sp. and dextran glucosidase from Streptococcus mutans. The DexG has four conserved regions shared with aamylases. In the cloned genomic fragment there were two other open reading frames, of which the deduced amino acid sequences showed a similarity with those of oligosaccharides membrane transporter proteins. The gene cluster consisting of the membrane transporter protein genes, dexG, and dexT, seems to participate in the degradation and utilization of dextran in this bacterium.

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 enzyme's 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.

Developing seeds of the kidney bean (Phaseolus vulgaris L.) contain several isoforms of starch branching enzymes. Two of them, KBE1 and KBE2, which are the major forms in the premature seeds, were purified as a single band of protein on SDS-PAGE and native PAGE by chromatographies on DEAE-Sepharose, Bio-Gel P-200, and amylose-binding Sepharose 6B. The enzymes had similar pH optimum (7.0), pH stability (7.0-9.5), temperature optimum (25-30 degrees C), and temperature stability (up to 40 degrees C). Additionally, both were inhibited by various divalent metal ions and activated by citrate. Finally, though their N-terminal amino acid sequences were identical, their molecular masses and affinities for amylose differed; 80 kDa and 1.27 mM for KBE1 and 77 kDa and 0.74 mM for KBE2.

A plant hormone, ethylene, is formed through 1-aminocyclopropane-1-carboxylic acid (ACC). A fungus, Penicillium citrium, was found to synthesize ACC and to degrade ACC into 2-oxobutyrate and ammonia. ACC synthase, responsible for ACC synthesis in P. citrinum, was characterized on the molecular level by sequencing of N terminal and proteolytic peptides of the enzyme, and cloning and sequencing of its cDNA. The ACC synthase from P. citrinum had 430 amino acid residues and a shorter C terminal than the plant enzyme. The enzyme purified from Escherichia coli transformed with ACC-synthase-encoding DNA showed similar properties to those of the purified enzyme from P. citrinum. Saccharomyces cerevisiae with ACC synthase accumulated ACC in the medium with increasing time of incubation. The sequence of ACC synthase from P. citrinum was compared with that of the plant enzyme with discussion about important residues for catalysis.

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.

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.