Hiromichi Minami

Functional Analysis of Norcoclaurine Synthase in Coptis japonica

(S)-Norcoclaurine is the entry compound in benzylisoquinoline alkaloid biosynthesis and is produced by the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) by norcoclaurine synthase (NCS) (EC 4.2.1.78). Although cDNA of the pathogenesis-related (PR) 10 family, the translation product of which catalyzes NCS reaction, has been isolated from Thalictrum flavum, its detailed enzymological properties have not yet been characterized. We report here that a distinct cDNA isolated from Coptis japonica (CjNCS1) also catalyzed NCS reaction as well as a PR10 homologue of C. japonica (CjPR10A). Both recombinant proteins stereo-specifically produced (S)-norcoclaurine by the condensation of dopamine and 4-HPAA. Because a CjNCS1 cDNA that encoded 352 amino acids showed sequence similarity to 2-oxoglutarate-dependent dioxygenases of plant origin, we characterized the properties of the native enzyme. Sequence analysis indicated that CjNCS1 only contained a Fe2+-binding site and lacked the 2-oxoglutarate-binding domain. In fact, NCS reaction of native NCS isolated from cultured C. japonica cells did not depend on 2-oxoglutarate or oxygen, but did require ferrous ion. On the other hand, CjPR10A showed no specific motif. The addition of o-phenanthroline inhibited NCS reaction of both native NCS and recombinant CjNCS1, but not that of CjPR10A. In addition, native NCS and recombinant CjNCS1 accepted phenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde, as well as 4-HPAA, for condensation with dopamine, whereas recombinant CjPR10A could use 4-hydroxyphenylpyruvate and pyruvate in addition to the above aldehydes. These results suggested that CjNCS1 is the major NCS in C. japonica, whereas native NCS extracted from cultured C. japonica cells was more active and formed a larger complex compared with recombinant CjNCS1.

Salt-tolerant γ-glutamyltranspeptidase from Bacillus subtilis 168 with glutaminase activity

γ-Glutamyltranspeptidase (GGT) from Bacillus subtilis is an extracellular enzyme that exhibits glutaminase activity and is thus suitable for the fermentation of foods. As GGT of B. subtilis is synthesized only during the mid-stationary phase, which is inconvenient for industrial use, a strain overexpressing GGT for a sufficiently long period was generated to obtain large quantities of GGT. A plasmid vector, pHY300PLK, containing the ggt gene cloned from chromosomal DNA was introduced into a spo0A abrB double mutant strain, in which the level of GGT activity is high after the mid-stationary phase. The level of GGT activity in this strain increased steadily after the exponential phase, becoming 15-fold higher than that in the parental strain. The recombinant GGT was purified by 252-fold with a yield of 30.4%. The enzyme is a heterodimer consisting of one large subunit (45 kDa) and one small subunit (21 kDa). The enzyme is highly salt-tolerant and converts glutamine to glutamic acid effectively even in the presence of 18% NaCl. This is the first report of salt-tolerant GGT.

A mutant Bacillus subtilisγ-glutamyltranspeptidase specialized in hydrolysis activity

γ-Glutamyltranspeptidase (GGT) catalyzes the hydrolysis of γ-glutamyl compounds and the transfer of their γ-glutamyl moieties to amino acids and peptides. The transpeptidation activity of Bacillus subtilis GGT is about 10-fold higher than its hydrolysis activity. In B. subtilis GGT, substitution of Asp-445 with Ala abolished its transpeptidation activity. The specific activity for hydrolysis of D445A GGT was 40.2% of that of the wild-type GGT. The K m value for l-glutamine was 15.3 mM. D445A GGT was salt tolerant like the wild-type GGT. These results indicate that D445A GGT will be highly useful as a ‘glutaminase’ in food industry. * Gly-Gly : glycylglycine γ-GCNA : γ-glutamyl-3-carboxy-4-nitroanilide γ-G p NA : γ-glutamyl- p -nitroanilide GGT : γ-glutamyltranspeptidase KPB : potassium phosphate buffer SDS : sodium dodecyl sulfate

Isolation of Herbicide-Resistant 4-Hydroxyphenylpyruvate Dioxygenase from Cultured Coptis japonica Cells

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the formation of homogentisate from 4-hydroxyphenylpyruvate and O2. In plants, HPPD has been identified as a molecular target for herbicides. We report the isolation and characterization of a cDNA encoding a HPPD from cultured Coptis japonica cells. Recombinant CjHPPD showed significantly higher half-maximum inhibitory concentration (IC50) values for the HPPD-inhibiting herbicide destosyl pyrazolate than other plant HPPDs.