Kazushi Koga

Streptomyces phospholipase D mutants with altered substrate specificity capable of phosphatidylinositol synthesis.

The substrate specificity of a phospholipase D (PLD) from Streptomyces antibioticus was altered by site‐directed saturation mutagenesis, so that it was able to synthesize phosphatidylinositol (PI). Mutations were introduced in the pld gene at the positions corresponding to three amino acid residues that might be involved in substrate recognition, and the mutated genes were expressed in Escherichia coli BL21 (DE3). High‐throughput screening of approximately 10 000 colonies for PI‐synthesizing activity identified 25 PI‐synthesizing mutant PLDs. One of these mutant enzymes was chosen for further analysis. The structure of the PI synthesized with the mutant enzyme was analyzed by HPLC‐MS and NMR. It was found that the mutant enzyme generated a mixture of structural isomers of PIs with the phosphatidyl groups connected at different positions of the inositol ring. The phosphatidylcholine‐hydrolyzing activity of the mutant PLD was much lower than that of the wild‐type enzyme. The mutant enzyme was able to transphosphatidylate various cyclohexanols with a preference for bulkier compounds. This is the first example of alteration of the substrate specificity of PLD and of PI synthesis by Streptomyces PLD.

First finding of Daidzein 7-O-phosphate and Genistein 7-O-phosphate that are hydrolyzed by sulfatase

Abstract Attempted structural assignment of two water soluble isoflavone analogs of Daidzein and Genistein, was initially assumed to be the corresponding sulfates on the basis of the facts that these analogs were hydrolyzed by sulfatase; however, they were eventually determined through NMR technology including 31P–13C couplings to be the corresponding phosphates both at the 7-O-position; thus, Daidzein 7-O-phosphate [4H-1-benzopyran-4-one,7-hydroxy-3-(4-hydroxyphenyl)-, 7-phosphate] and Genistein 7-O-phosphate [4H-1-benzopyran-4-one, 5,7 –dihydroxy-3-(4-hydroxyphenyl)-, 7-phosphate].

Complexation of cyclic dodecadepsipeptide, cereulide with ammonium salts.

Cereulide is a principal toxin causing emetic syndrome which is produced by Bacillus cereus and has been known as potassium selective ionophore. This paper deals with its complexation with inorganic and organic ammonium ions to assign the higher structures similar to the complex with potassium ion by means of NMR and ESI-MS spectroscopy. Of particular interest, the detectable ions are not only at m/z 1191.8 as K(+) complex but also (or sometimes exclusively) at m/z 1170.8 as NH(4)(+) complex in its LC-MS analyses depending upon the conditions. This difference is due to the sample preparation and measurement condition.

7,8-Dihydropterin-6-carboxylic Acid as Light Emitter of Luminous Millipede, Luminodesmus sequoiae

A luminous millipede. Luminodesmus sequoiae, emits light centered at a wavelength of 500 nm. To determine the light emitter of this bioluminescent system, fluorescent compounds were isolated from pulverized cuticles. NMR and MS spectra of these compounds showed them to be pterin derivatives. Furthermore, proton/deuterium (H/D) exchange experiments by ESI-Q-TOF-MS and -MS/MS measurements have proved to be a powerful tool for elucidating these heteroaromatic compounds. Finally, we have concluded that 7,8-dihydropterin-6-carboxylic acid, a new natural product, is the light emitter of Luminodesmus bioluminescence.

High incorporation of l-amino acids to cereulide, an emetic toxin from Bacillus cereus

Cereulide is a principal toxin causing emetic syndrome produced by Bacillus cereus. This paper deals with biosynthetic studies on this unusual cyclic depsipeptide toxin from 13C labeled L-amino acid precursors (Val, Leu, Ala) upon cultivation in synthetic media. The analyses were made at atomic level of the constituent amino- or oxy-acids through NMR and ESI-MS/MS spectroscopic methods on cereulide and its hydrolysate dipeptides. The incorporation of the 13C atom was 95% in each O-Val, O-Leu and L-Val, while 40% in D-Ala of cereulide.

19F-Dehydrocoelenterazine as probe to investigate the active site of symplectin

Abstract Fluorinated dehydrocoelenterazines (F-DCTs) were synthesized to study molecular mechanisms of symplectin; a photoprotein of luminous squid Symplectoteuthis oualaniensis L. F-DCTs reacted with dithiothreitol and glutathione under neutral conditions to give the stable chromophores as symplectin model. Reconstructed symplectin was also obtained by addition of F-DCTs into apo-symplectin, and showed bioluminescence to emit 50–65% amount of light as natural symplectin. The structure of the chromophores was determined by 19F NMR, Q-TOF-MS, and MS/MS analyses. Sequencing of the chromopeptides of symplectin models prepared from F-DCTs and thiol compounds was accomplished by ESI-Q-TOF-MS/MS analysis.

Role of zinc ion for catalytic activity in d-serine dehydratase from Saccharomyces cerevisiae

d‐Serine dehydratase from Saccharomyces cerevisiae (DsdSC) is a fold‐type III pyridoxal 5′‐phosphate‐dependent enzyme catalyzing d‐serine dehydration. The enzyme contains 1 mol Zn2+ in its active site and shows a unique zinc dependence. The Zn2+ is essential for the d‐serine dehydration, but not for the α,β‐elimination of β‐Cl‐d‐alanine catalyzed as a side‐reaction. The fact that dehydration of d‐threonine and d‐allo‐threonine, also catalyzed by DsdSC, is likewise Zn2+ dependent indicates that Zn2+ is indispensable for the elimination of hydroxyl group, regardless of the stereochemistry of Cβ. Removal of Zn2+ results in a less polar active site without changing the gross conformation of DsdSC. 1H NMR determined the rates of α‐hydrogen abstraction and hydroxyl group elimination of d‐serine in 2H2O to be 9.7 and 8.5 s−1, respectively, while the removal of Zn2+ abolished both reactions. Mutation of Cys400 or His398 within the Zn2+ binding sites to Ala endowed DsdSC with similar properties to those of the Zn2+‐depleted wild‐type enzyme: the mutants lost the reactivity toward d‐serine and d‐threonine but retained that toward β‐Cl‐d‐alanine. 1H NMR analysis also revealed that both α‐hydrogen abstraction and hydroxyl group elimination from d‐serine were severely hampered in the C400A mutant. Our data suggest that DsdSC catalyzes the α‐hydrogen abstraction and hydroxyl group elimination in a concerted fashion.

A cis-prenyltransferase from Methanosarcina acetivorans catalyzes both head-to-tail and nonhead-to-tail prenyl condensation.

Cis‐prenyltransferase usually consecutively catalyzes the head‐to‐tail condensation reactions of isopentenyl diphosphate to allylic prenyl diphosphate in the production of (E,Z‐mixed) polyprenyl diphosphate, which is the precursor of glycosyl carrier lipids. Some recently discovered homologs of the enzyme, however, catalyze the nonhead‐to‐tail condensation reactions between allylic prenyl diphosphates. In this study, we characterize a cis‐prenyltransferase homolog from a methanogenic archaeon, Methanosarcina acetivorans, to obtain information on the biosynthesis of the glycosyl carrier lipids within it. This enzyme catalyzes both head‐to‐tail and nonhead‐to‐tail condensation reactions. The kinetic analysis shows that the main reaction of the enzyme is consecutive head‐to‐tail prenyl condensation reactions yielding polyprenyl diphosphates, while the chain lengths of the major products seem shorter than expected for the precursor of glycosyl carrier lipids. On the other hand, a subsidiary reaction of the enzyme, i.e., nonhead‐to‐tail condensation between dimethylallyl diphosphate and farnesyl diphosphate, gives a novel diterpenoid compound, geranyllavandulyl diphosphate.

Structural Study on a Naturally Occurring Terphenyl Quinone

Two terphenyl quinones were synthesized for a structural study on a naturally occurring biologically active terphenyl quinone. 3-Methoxy-5,6-diphenylcyclohexa-3,5-dien-1,2-dione, a possible structure proposed by our analysis of the NMR spectra, was synthesized by Suzuki-Miyaura coupling and subsequent oxidation of the resulting substituted phenol, although not being identical to the natural product. Recently isolated 3-methoxy-2,5-diphenylcyclohexa-2,5-dien-1,4-dione was synthesized from a commercially available 2,5-diphenyl-1,4-benzoquinone in three steps in a good overall yield, and its NMR spectra were identical to those of the natural product.