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Journal of Biochemistry Advance Access published online on October 27, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp165
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Topology of 4-Pyridoxic Acid Dehydrogenase in Transformed Escherichia coli Cells

Fei Ge1 and Toshiharu Yagi1,*

1 Department of Bioresources Science, Faculty of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan

*To whom correspondence should be addressed: Toshiharu Yagi Tel and Fax: +81-88-864-5191; E-mail: yagito{at}kochi-u.ac.jp

Received September 26, 2009; Accepted October 7, 2009


  Abstract

The topology of 4-pyridoxic acid dehydrogenase in the Escherichia coli cell membrane was examined with transformed E. coli cells overexpressing the enzyme from Mesorhizobium loti. The recombinant enzymes with a His6-tag on N-terminal region or C-terminal were localized on the E. coli cell membrane like the wild-type enzyme without a His6-tag. The His6-tags were labeled with Ni-NTA AP conjugate only when the E. coli protoplast cells were broken. The membrane-bound enzyme in the intact protoplast cells was not digested by trypsin, although the one in the gently broken protoplast cells was almost totally digested. Thus, 4-pyridoxic acid dehydrogenase was an integral monotopic protein, protruding into a cytoplasm side from the bacterial membrane. The deletion or mutation of a deduced transmembrane segment in 4-pyridoxic acid dehydrogenase made it an inclusion body, and the enzyme protein was not found in the E. coli cell membrane. Thus, it was suggested that the intact deduced transmembrane segment was necessary for 4-pyridoxic acid dehydrogenase to be localized on the bacterial cell membrane.

Key Words: membrane topology, 4-pyridoxic acid dehydrogenase, pyridoxine (vitamin B6)-degradation, Mesorhizobium loti


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