A
cDNA encoding cabbage
histidinol dehydrogenase, including the chloroplast transit
peptide sequence, was overexpressed using a baculovirus expression vector system. The maximum level of the expression of
histidinol dehydrogenase was reached 5 days after
infection of the insect cells. Two forms of recombinant
histidinol dehydrogenase with molecular masses of 53 and 52 kDa, respectively, were obtained by a one-step purification from the cell homogenate. Compared with the 52-kDa form, the 53-kDa form contained 10 additional
amino acids at the N-terminus derived from the transit
peptide. By incubating the cell homogenate for 2 h at 30 degrees C, the 53-kDa form could be completely converted to the 52-kDa form. This conversion was blocked by
leupeptin. Eighty percent of the converted 52-kDa form had Cys at position 31 at the N-terminal
amino acid and the rest had Met 33. Kinetic properties of the recombinant
enzyme were virtually identical to those of
histidinol dehydrogenase isolated from cabbage plants. The overexpression of recombinant cabbage
histidinol dehydrogenase in insect cells, the proteolytic processing of the preprotein next to the N-terminus (compared to the mature cabbage
enzyme), and its easy purification allow the preparation of large amounts of the active
enzyme for structural and functional studies.