Enzymatic Synthesis of the Crithidia Factors in Cell-free Extracts of Serratia indica

The concomitant production of formic acid and pterin compounds from guanosine-5′-triphosphate (GTP) has been found in cell-free extracts of Serratia indica. Among the pterin compounds, l-threo-neopterin–the major Crithidia factor in S. indica–, a cyclic phosphate of neopterin (cNP), d-erythro-neopterin and 6-hydroxymethyl pterin were detected and isolated. Formate-¹⁴C elimination from GTP-8-¹⁴C was quantitatively distributed in the ethyl acetate layer in the ehyl acetate-hydrochloric acid partition system. Carbon 8 of GTP was released as formic acid. Enzymatic production of formate and cNP was linear for 2 hr at 37°C. Formate production was proportional to the enzyme concentration. The optimum pH for formate elimination was observed around pH 8.6. Optimum temperature for the production of formate and cNP was 50°C. The apparent Km value of GTP for formate production was 6.2×10^?bm. Formate eliminating activity was activated by disodium phosphate but was inhibited by Mg²⁺ or AMP. Incorporation of GTP-U-¹⁴C into pterin compounds was also regulated with disodium phosphate. Effective incorporation into cNP and d-erythro-neopterin occurred in the presence of phosphate. When phosphate was omitted from the system, however, effective incorporation into 6-hydroxymethyl pterin was observed. The biosynthetic process of the Crithidia factors, i.e. l-threo-neopterin and cNP, from GTP in S. indica is also discussed.     

Comparison of Mineral Balances in Germfree and Conventional Mice When Sodium Phytate is Added to Purified Diet

A strain of Alcaligenes isolated from soil was a good producer of β-glucuronidase, and the enzyme was purified from the cell-free extract by sequential column chromatography on DEAE-Toyopearl, Toyopearl HW-55F, and Phenyl-Sepharose CL-4B. By these procedures, two β-glucuronidases designated as β-glucuronidases I and II were purified 240- and 508-fold, respectively. β-Glucuronidase I, with a molecular weight of 75,000, had an optimum pH at 7.5 and the enzyme II, with a molecular weight of 300,000, had maximum activity at pH 6.0. Both enzymes were strongly inhibited by saccharo-1,4-lactone, glucaro-δ-lactam, p-chloromercuribenzoate, Hg²⁺, and N-bromosuccinimide. β-Glucuronidase I was active toward estrogen-3-β-glucuronides and inert toward β-glucuronide conjugates of menthol, estrogen-17β-, estrogen-16α-, androsterone-3α-, testosterone-17β-, cortisol-17α-. β-Glucuronidase II hydrolyzed all of these substrates. β-Glucuronidase I was inhibited by phenolphthalein and its glucuronide.     

Intracellular Distributions of Enzymes and Intermediates Involved in Biosynthesis of Capsaicin and Its Analogues in Capsicum Fruits

Phenylalanine ammonia-lyase, trans-cinnamate 4-monooxygenase, and capsaicinoid synthetase [Agric. Biol. Chem., 44, 2907 (1980)] activities were investigated in the subcellular fractions from protoplasts of placenta of Capsicum fruits. The subcellular distribution of intermediates of the capsaicinoid biosynthesis, trans-cinnamic acid and trans-p-coumaric acid, and capsaicinoid were also investigated. The activity of trans-cinnamate 4-monooxygenase and capsaicinoid synthetase was in the vacuole fraction. While the activity of phenylalanine ammonia-lyase was in the cytosol fraction. After feeding l-[U-¹⁴C]phenylalanine to the protoplast, the newly synthesized trans-p-coumaric acid and capsaicinoid were found in the vacuole fraction, while trans-cinnamic acid was not in the vacuole fraction. The possible role of the vacuole on the biosynthesis of capsaicinoid is also discussed.     

Identification of d-Erythro-Dihydroneopterin Triphosphate as a Product of Guanosine Triphosphate Cyclohydrolase from Serratia indica

Guanosine triphosphate cyclohydrolase (EC 3.5.4.16) was previously shown to exist in two forms (GTP cyclohydrolase D-I and D-II) in Serratia indica IFO 3759, and they were homogeneously isolated. The present study deals with the characterization of their reaction products. A fluorescent product formed from guanosine triphosphate by GTP cyclohydrolase D-II was identified as 7,8-dihydroneopterin triphosphate by its absorption spectra, phosphate analysis and gas chromatography-mass spectrometry of the dephosphorylated trimethylsilyl derivative. After oxidation and dephosphorylation, the d-erythro configuration of the side chain was made clear by the elution profile on ECTEOLA-cellulose chromatography, Rf values on thin-layer chromatography and by biological activity to Crithidia fasciculata ATCC 12857. The fluorescent products from GTP cyclohydrolase D-I and D-II were indistinguishable.     

Formation of Pungent Principles in Fruits of Sweet Pepper,Capsicum annuum L. var. grossum during Post-harvest Ripening under Continuous Light

Pungent principles (Capsaicinoid(s)) were found to be produced in fruits of sweet pepper, Capsicum annuum L. var. grossum, during post-harvest ripening under continuous light. The initial formation was observed after 4 days’ ripening. After 7 days’ ripening, the capsaicinoids content in placenta increased to 12.9 μg per fruit, which was 2.5-fold of that in pericarp. No pungent principles were detected in fruits during ripening in the dark and in seeds under continuous light. In placenta, the formation of dihydrocapsaicin and nordihydrocapsaicin which are the vanillylamides of saturated branched fatty acids was higher than that of capsaicin which is the vanillylamide of an unsaturated one. Remarkable formation and accumulation of carotenoid were also observed during post-harvest ripening under continuous light.     

Properties of the Crystalline Quinolinate Phosphoribosyltransferase from Hog Liver

Quinolinate phosphoribosyltransferase has an important role in the NAD de novo biosynthetic pathway. Crystalline quinolinate phosphoribosyltransferase could be obtained for the first time from mammalian tissue. The crystalline enzyme preparation was certified to be homogeneous by polyacrylamide gel disc electrophoresis. Catalytic properties of this enzyme preparation were investigated. Optimum pH for the reaction was 6.1. Divalent cations were absolutely required and Mg²⁺ was the most effective. Michaelis constants for quinolinic acid and PRPP were 1.2 × 10^?4 m and 1.8 × 10^?4 m, respectively. Quinolinic acid could not be replaced by nicotinic acid or 2-amino nicotinic acid in this reaction. Di- and tri-valent cations fairly inhibited the reaction, but mono-valent cations had no effects. The reaction product was identified as β-nicotinic acid mononucleotide by its ultraviolet absorption spectra, paper chromatography, paper electrophoresis and its ORD spectrum.     

Mutation strategies for obtaining chitooligosaccharides with longer chains by transglycosylation reaction of family GH18 chitinase

Enhancing the transglycosylation (TG) activity of glycoside hydrolases does not always result in the production of oligosaccharides with longer chains, because the TG products are often decomposed into shorter oligosaccharides. Here, we investigated the mutation strategies for obtaining chitooligosaccharides with longer chains by means of TG reaction catalyzed by family GH18 chitinase A from Vibrio harveyi (VhChiA). HPLC analysis of the TG products from incubation of chitooligosaccharide substrates, GlcNAc_n, with several mutant VhChiAs suggested that mutant W570G (mutation of Trp570 to Gly) and mutant D392N (mutation of Asp392 to Asn) significantly enhanced TG activity, but the TG products were immediately hydrolyzed into shorter GlcNAc_n. On the other hand, the TG products obtained from mutants D313A and D313N (mutations of Asp313 to Ala and Asn, respectively) were not further hydrolyzed, leading to the accumulation of oligosaccharides with longer chains. The data obtained from the mutant VhChiAs suggested that mutations of Asp313, the middle aspartic acid residue of the DxDxE catalytic motif, to Ala and Asn are most effective for obtaining chitooligosaccharides with longer chains.     

Purification,cDNA cloning,and characterization of LysM-containing plant chitinase from horsetail (Equisetum arvense)

Chitinase-A (EaChiA), molecular mass 36 kDa, was purified from the vegetative stems of a horsetail (Equisetum arvense) using a series of column chromatography. The N-terminal amino acid sequence of EaChiA was similar to the lysin motif (LysM). A cDNA encoding EaChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1320 nucleotides and encoded an open reading frame of 361 amino acid residues. The deduced amino acid sequence indicated that EaChiA is composed of a N-terminal LysM domain and a C-terminal plant class IIIb chitinase catalytic domain, belonging to the glycoside hydrolase family 18, linked by proline-rich regions. EaChiA has strong chitin-binding activity, however, no antifungal activity. This is the first report of a chitinase from Equisetopsida, a class of fern plants, and the second report of a LysM-containing chitinase from a plant.