All of the enzyme activities, with the exception of pyridoxine 4-

All of the enzyme activities, with the exception of pyridoxine 4-oxidase (step 1a), of the disruptant strain grown in TY medium were significantly increased

compared with the wild-type strain; pyridoxal 4-dehydrogenase showed the highest specific activity of 300 nmol min−1 mg−1 protein (about 31-fold higher than that in the wild-type cells). The results showed that the PyrR-disruptant constitutively expressed the eight Dabrafenib enzymes of the pyridoxine degradation pathway and that PyrR was a repressor. The wild-type cells grown in PN synthetic medium showed significantly higher enzyme activities than those grown in TY medium because the degradation pathway is induced in this medium (Guirard & Snell, 1971). When the disruptant cells were grown in PN medium, the activities of the enzymes catalyzing steps 1b, 3, and 6 of the degradation pathway selleckchem were found to be significantly higher than those in cells grown in TY medium. In contrast, the activities of the enzymes catalyzing steps 4 and 5 were significantly lower than those in cells grown in TY medium. The activities of the other enzymes in the disruptant cells were almost the same regardless of the medium. As described

below, because the genes mlr6792 and mlr6793 encoding the enzymes of step 4 and step 5, respectively, two genes may constitute an operon, the same pattern of changes in the enzyme activities may be rational. However, the results do suggest that some factor(s) in addition to the PyrR protein contribute to control of synthesis of the enzymes. A crude extract of E. coli cells transformed with pET6786-His6 gave a dense protein band corresponding to the molecular mass of PyrR (25 000 Da) on an SDS-PAGE gel (Fig. 2b). Thus, PyrR appeared to have been cloned and expressed in the E. coli cells. PyrR-His6 was purified by Ni-NTA-affinity chromatography (Fig. 2b). The molecular mass of the intact PyrR-His6 was determined to be 50 000 Da by size-exclusion chromatography (data not shown), showing that it is a dimeric

protein like other VanR family proteins. Three DNA fragments were prepared and labeled with biotin (Fig. 3a) and the interaction of the DNA fragments with PyrR was then examined. It was found that the 321-bp and 135-bp fragments bound to PyrR, and their movements on the polyacrylamide gel were affected (Fig. 3b and c). In contrast, a 68-bp mafosfamide fragment did not bind to PyrR and its movement was not affected (Fig. 3d). The results showed that the PyrR protein bound to an intergenic 67-bp DNA region (Fig. 3a). The 67-bp DNA contains a palindrome sequence (GATTGTCAGACAATC). It has been reported that E. coli FadR binds to a palindrome sequence (TGGTCCGACCA or TGGTACGACCA; Xu et al., 2001). Thus, PyrR may bind to the palindrome sequence in the 67-bp DNA and inhibit the expression of the mlr6787 operon. The palindrome sequence overlapped the predicted –10 sequence of a putative promoter for the mlr6787 gene.

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