Tokuzo Nishino

A Role of the Amino Acid Residue Located on the Fifth Position before the First Aspartate-rich Motif of Farnesyl Diphosphate Synthase on Determination of the Final Product

Farnesyl diphosphate (FPP) synthase catalyzes consecutive condensations of isopentenyl diphosphate with allylic substrates to give FPP, C-15 compound, as a final product and does not catalyze a condensation beyond FPP. Recently, it was observed that, in Bacillus stearothermophilus FPP synthase, a replacement of tyrosine with histidine at position 81, which is located on the fifth amino acid before the first aspartate-rich motif, caused the mutated FPP synthase to catalyze geranylgeranyl diphosphate (C-20) synthesis (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T. (1996) J. Biol. Chem. 271, 10087-10095). Thus, we constructed 20 FPP synthases, each of which has a different amino acid at position 81, and analyzed them. All enzymes except for Y81P can catalyze the condensations of isopentenyl diphosphate. The final products and the product distributions are different from each other. Y81A, Y81G, and Y81S can produce hexaprenyl diphosphate (C-30) as their final product. The final product of Y81C, Y81H, Y81I, Y81L, Y81N, Y81T, and Y81V are geranylfarnesyl diphosphate (C-25), and Y81D, Y81E, Y81F, Y81K, Y81M, Y81Q, and Y81R cannot produce polyprenyl diphosphates more than geranylgeranyl diphosphate. Substitution of tryptophan does not affect the product specificity of FPP synthase. The average chain length of products is inversely proportional to the accessible surface area of substituted amino acid. However, no significant relation between the final chain length and the kinetic constants Km and Vmax are observed. These observations strongly indicate that the amino acid does not come into contact with the substrates but directly contacts the ω-terminal of an elongating allylic product. This interaction must prevent further condensation of isopentenyl diphosphate.

Conversion of Product Specificity of Archaebacterial Geranylgeranyl-diphosphate Synthase IDENTIFICATION OF ESSENTIAL AMINO ACID RESIDUES FOR CHAIN LENGTH DETERMINATION OF PRENYLTRANSFERASE REACTION

Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. To investigate the mechanism of the consecutive reaction and the determination of the ultimate chain length, a random mutational approach was planned. A geranylgeranyl-diphosphate synthase gene from Sulfolobus acidocaldarius was randomly mutagenized by NaNO2 treatment to construct a library of mutated geranylgeranyl-diphosphate synthase genes on a yeast expression vector. The library was screened for suppression of a pet phenotype of yeast C296-LH3, which is deficient in hexaprenyl-diphosphate synthase. Five mutants that could grow on a YEPG plate, which contained only glycerol as an energy source instead of glucose, were selected from ~1,400 mutants. All selected mutated enzymes catalyzed the formation of polyprenyl diphosphates with prenyl chains longer than geranylgeranyl diphosphate. Especially mutants 1, 3, and 5 showed the strongest elongation activity to produce large amounts of geranylfarnesyl diphosphate with a concomitant amount of hexaprenyl diphosphate. Sequence analysis revealed that each mutant contained a few amino acid substitutions and that the mutation of Phe-77, which is located on the fifth amino acid upstream from the first aspartate-rich consensus motif, is the most effective for elongating the ultimate product. Amino acid alignment of known prenyltransferases around this position and our previous observations on farnesyl-diphosphate synthase (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T. (1996) J. Biol. Chem. 271, 10087-10095) clearly indicate that the amino acid at the position of all prenyltransferases must regulate the chain elongation.

Polyprenyl diphosphate synthase essentially defines the length of the side chain of ubiquinone

Ubiquinone, known as a component of the electron transfer system in many organisms, has a different length of the isoprenoid side chain depending on the species, e.g., Escherichia coli, Saccharomyces cerevisiae and humans have 8, 6, and 10 isoprene units in the side chain, respectively. No direct evidence has yet shown what factors define the length of the side chain of ubiquinone. Here we proved that the polyprenyl diphosphate that was available in cells determined the length of the side chain of ubiquinone. E. coli octaprenyl diphosphate synthase (IspB) was expressed with the mitochondrial import signal in S. cerevisiae. Such cells produced ubiquinone-8 in addition to the originally existing ubiquinone-6. When IspB was expressed in a S. cerevisiae COQ1 defective strain. IspB complemented the defect of the growth on the non-fermentable carbon source. Those cells had the activity of octaprenyl diphosphate synthase and produced only ubiquinone-8. These results opened the possibility of producing the type of ubiquinone that we need in S. cerevisiae simply by expressing the corresponding polyprenyl diphosphate synthase.

Conversion from Archaeal Geranylgeranyl Diphosphate Synthase to Farnesyl Diphosphate Synthase TWO AMINO ACIDS BEFORE THE FIRST ASPARTATE-RICH MOTIF SOLELY DETERMINE EUKARYOTIC FARNESYL DIPHOSPHATE SYNTHASE ACTIVITY

Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are precursors for a variety of important natural products, such as sterols, carotenoids, and prenyl quinones. Although FPP synthase and GGPP synthase catalyze similar consecutive condensations of isopentenyl diphosphate with allylic diphosphates and have several homologous regions in their amino acid sequences, nothing is known about how these enzymes form the specific products. To locate the region that causes the difference of final products between GGPP synthase and FPP synthase, we constructed six mutated archaeal GGPP synthases whose regions around the first aspartate-rich motif were replaced with the corresponding regions of FPP synthases from human, rat, Arabidopsis thaliana, Saccharomyces cerevisiae, Escherichia coli, Bacillus stearothermophilus, and from some other related mutated enzymes. From the analysis of these mutated enzymes, we revealed that the region around the first aspartate-rich motif is essential for the product specificity of all FPP synthases and that the mechanism of the chain termination in eukaryotic FPP synthases (type I) is different from those of prokaryotic FPP synthases (type II). In FPP synthases of type I, two amino acids situated at the fourth and the fifth positions before the motif solely determine their product chain length, while the product specificity of the type II enzymes is determined by one aromatic amino acid at the fifth position before the motif, two amino acids inserted in the motif, and other modifications. These data indicate that FPP synthases have evolved from the progenitor corresponding to the archaeal GGPP synthase in two ways.

A UDP-Glucose:Isoflavone 7-O-Glucosyltransferase from the Roots of Soybean (Glycine max) Seedlings PURIFICATION, GENE CLONING, PHYLOGENETICS, AND AN IMPLICATION FOR AN ALTERNATIVE STRATEGY OF ENZYME CATALYSIS

Isoflavones, a class of flavonoids, play very important roles in plant-microbe interactions in certain legumes such as soybeans (Glycine max L. Merr.). G. max UDP-glucose:isoflavone 7-O-glucosyltransferase (GmIF7GT) is a key enzyme in the synthesis of isoflavone conjugates, which accumulate in large amounts in vacuoles and serve as an isoflavonoid pool that allows for interaction with microorganisms. In this study, the 14,000-fold purification of GmIF7GT from the roots of G. max seedlings was accomplished. The purified enzyme is a monomeric protein of 46 kDa, catalyzing regiospecific glucosyl transfer from UDP-glucose to isoflavones to produce isoflavone 7-O-β-d-glucosides (kcat = 0.74 s-1, Km for genistein = 3.6 μm, and Km for UDP-glucose = 190 μm). The GmIF7GT cDNA was isolated based on the amino acid sequence of the purified enzyme. Phylogenetic analysis showed that GmIF7GT is a novel member of glycosyltransferase family 1 and is distantly related to Glycyrrhiza echinata UDP-glucose:isoflavonoid 7-O-glucosyltransferase. The purified enzyme was unexpectedly devoid of the N-terminal 49-residue segment and thus lacks the histidine residue corresponding to the proposed catalytic residue of glycosyltransferases from Medicago truncatula (UGT71G1) and Vitis vinifera (VvGT1). The results of kinetic studies of site-directed mutants of GmIF7GT showed that both His-15 and Asp-125, which correspond to the catalytic residues of UGT71G1 and VvGT1, are not important for GmIF7GT activity. The results also suggest that an acidic residue at position 392 is very important for primary catalysis of GmIF7GT. These results led to the proposal that GmIF7GT utilizes a strategy of catalysis that is distinct from those proposed for UGT71G1 and VvGT1.

UDP-glucuronic acid: Anthocyanin glucuronosyltransferase from red daisy (Bellis perennis) flowers enzymology and phylogenetics of a novel glucuronosyltransferase involved in flower pigment biosynthesis

In contrast to the wealth of biochemical and genetic information on vertebrate glucuronosyltransferases (UGATs), only limited information is available on the role and phylogenetics of plant UGATs. Here we report on the purification, characterization, and cDNA cloning of a novel UGAT involved in the biosynthesis of flower pigments in the red daisy (Bellis perennis). The purified enzyme, BpUGAT, was a soluble monomeric enzyme with a molecular mass of 54 kDa and catalyzed the regiospecific transfer of a glucuronosyl unit from UDP-glucuronate to the 2″-hydroxyl group of the 3-glucosyl moiety of cyanidin 3-O-6″-O-malonylglucoside with a kcat value of 34 s–1 at pH 7.0 and 30 °C. BpUGAT was highlyspecific for cyanidin 3-O-glucosides (e.g. Km for cyanidin 3-O-6″-O-malonylglucoside, 19 μm) and UDP-glucuronate (Km, 476 μm). The BpUGAT cDNA was isolated on the basis of the amino acid sequence of the purified enzyme. Quantitative PCR analysis showed that transcripts of BpUGAT could be specifically detected in red petals, consistent with the temporal and spatial distributions of enzyme activity in the plant and also consistent with the role of the enzyme in pigment biosynthesis. A sequence analysis revealed that BpUGAT is related to the glycosyltransferase 1 (GT1) family of the glycosyltransferase superfamily (according to the Carbohydrate-Active Enzymes (CAZy) data base). Among GT1 family members that encompass vertebrate UGATs and plant secondary product glycosyltransferases, the highest sequence similarity was found with flavonoid rhamnosyltransferases of plants (28–40% identity). Although the biological role (pigment biosynthesis) and enzymatic properties of BpUGAT are significantly different from those of vertebrate UGATs, both of these UGATs share a similarity in that the products produced by these enzymes are more water-soluble, thus facilitating their accumulation in vacuoles (in BpUGAT) or their excretion from cells (in vertebrate UGATs), corroborating the proposed general significance of GT1 family members in the metabolism of small lipophilic molecules.

An Isoflavone Conjugate-hydrolyzing β-Glucosidase from the Roots of Soybean (Glycine max) Seedlings PURIFICATION, GENE CLONING, PHYLOGENETICS, AND CELLULAR LOCALIZATION

Soybeans (Glycine max (L.) Merr.) and certain other legumes excrete isoflavones from their roots, which participate in plantmicrobe interactions such as symbiosis and as a defense against infections by pathogens. In G. max, the release of free isoflavones from their conjugates, the latent forms, is mediated by an isoflavone conjugate-hydrolyzing β-glucosidase. Here we report on the purification and cDNA cloning of this important β-glucosidase from the roots of G. max seedlings as well as related phylogenetic and cellular localization studies. The purified enzyme, isoflavone conjugate-hydrolyzing β-glucosidase from roots of G. max seedling (GmICHG), is a homodimeric glycoprotein with a subunit molecular mass of 58 kDa and is capable of directly hydrolyzing genistein 7-O-(6 ″-O-malonyl-β-d-glucoside) to produce free genistein (kcat, 98 s-1; Km, 25 μm at 30 °C, pH 7.0). GmICHG cDNA was isolated based on the amino acid sequence of the purified enzyme. GmICHG cDNA was abundantly expressed in the roots of G. max seedlings but only negligibly in the hypocotyl and cotyledon. An immunocytochemical analysis using anti-GmICHG antibodies, along with green fluorescent protein imaging analyses of Arabidopsis cultured cells transformed by the GmICHG:GFP fusion gene, revealed that the enzyme is exclusively localized in the cell wall and intercellular space of seedling roots, particularly in the cell wall of root hairs. A phylogenetic analysis revealed that GmICHG is a member of glycoside hydrolase family 1 and can be co-clustered with many other leguminous β-glucosidases, the majority of which may also be involved in flavonoid-mediated interactions of legumes with microbes.

Specificity analysis and mechanism of aurone synthesis catalyzed by aureusidin synthase, a polyphenol oxidase homolog responsible for flower coloration.

Aureusidin synthase, which plays a key role in the yellow coloration of snapdragon flowers, is a homolog of plant polyphenol oxidase (PPO). The enzyme specifically acted on chalcones with a 4‐monohydroxy or 3,4‐dihydroxy B‐ring to produce aurones, for whose production the oxidative cyclization of chalcones must be preceded by 3‐oxygenation. However, it exhibited virtually no PPO activity toward non‐chalcone phenolics. The enzyme was competitively inhibited by phenylthiourea, a specific PPO inhibitor. These results led us to propose a mechanism of aurone synthesis by aureusidin synthase on the basis of known PPO‐catalyzed reactions and conclude that the enzyme is a chalcone‐specific PPO specialized for aurone biosynthesis.

A pathway where polyprenyl diphosphate elongates in prenyltransferase. Insight into a common mechanism of chain length determination of prenyltransferases

Prenyltransferases catalyze the consecutive condensations of isopentenyl diphosphate to produce linear polyprenyl diphosphates. Each enzyme forms the final product with a specific chain length. The product specificity of an enzyme is thought to be determined by the structure around the unknown path through which the product elongates in the enzyme. To explore the path, we introduced a few mutations at the 5th, the 8th, and/or the 11th positions before the first aspartate-rich motif of geranylgeranyl-diphosphate synthase or farnesyl-diphosphate synthase. The side chains of these amino acids are situated on the same side of an α-helix. In geranylgeranyl-diphosphate synthase, a single mutated enzyme (F77S) mainly produces a C25 product (Ohnuma, S.-I., Hirooka, K., Hemmi, H., Ishida, C., Ohto, C., and Nishino, T. (1996)J. Biol. Chem. 271, 18831–18837). A double mutated enzyme (L74G and F77G) mainly produces a C35 compound with significant amounts of C30 and C40. A triple mutated enzyme (I71G, L74G, and F77G) mainly produces a C40compound with C35 and C45. Mutated farnesyl-diphosphate synthases also show similar patterns. These findings indicate that the elongating product passages on a surface of the side chains of the mutated amino acids, the original bulky amino acids had blocked the elongation, and the path is conserved in prenyltransferases. Moreover, the fact that some double and triple mutated enzymes can also form small amounts of products longer than C50 indicates that the paths in these mutated enzymes can partially access the outer surface of the enzymes.

Cold-active lipolytic activity of psychrotrophic Acinetobacter sp. strain No. 6

A lipolytic bacterium, strain no. 6, was isolated from Siberian tundra soil. It was a gram-negative coccoid rod capable of growing at 4 degrees C but not at 37 degrees C and was identified as a psychrotrophic strain of the genus Acinetobacter. Strain no. 6 extracellularly produced a lipolytic enzyme that efficiently hydrolyzed triglycerides such as soybean oil during bacterial growth even at 4 degrees C; it degraded 60% of added soybean oil (initial concentration, 1% w/v) after cultivation in LB medium at 4 degrees C for 7 d. Thus, the bacterium is potentially applicable to in-situ bioremediation or bioaugumentation of fat-contaminated cold environments. We partially purified the lipolytic enzyme from the culture filtrate by acetone fractionation and characterized it. The enzyme preparation contained a single species of cold-active lipase with significant activity at 4 degrees C, which was 57% of the activity at the optimum temperature (20 degrees C). The enzyme showed a broad specificity toward the acyl group (C8-C16) of substrate ethyl esters.