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The partial amino acid sequence of histidinol dehydrogenase (L-histidinol:NAD+ oxidoreductase, EC 1.1.1.23) from cabbage was determined from peptide fragments of the purified protein. The relative positions of these peptides were deduced by aligning their sequences with the sequence of the HIS4C gene product of Saccharomyces cerevisiae. cDNA encoding histidinol dehydrogenase was then amplified from a library using a polymerase chain reaction primed with degenerate oligonucleotide pools of known position and orientation. By using this amplified fragment as a probe, an apparently fulD-length cDNA clone was isolated that is predicted to encode a proenzyme having a putative 31-amino acid chloroplast transit peptide and a mature molecular mass of47.5 kDa. The predicted protein sequence was 51% identical to the yeast enzyme and 49% identical to the Escherichia coli enzyme. Expression of the cDNA clone in an E. coli his operon deletion strain rendered the mutant able to grow in the presence of histidinol.In plants, the biosynthetic pathways of most of the amino acids are poorly understood. Only a handful of enzymes involved in amino acid biosynthesis have been purified from plant sources, partly because of the small amount of these proteins found in plant cells. To date, glutamate synthase, glutamate dehydrogenase, and glutamine synthetase in the glutamate pathway (1-3), aspartate kinase (4) and homoserine dehydrogenase (5) in the threonine pathway, dihydrodipicolinate synthase (6) in the lysine pathway, and 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (7) and 5-enoylpyruvyl shikimate-3-phosphate synthase (8) in the aromatic amino acid pathway have been characterized and purified from various plant species. cDNAs encoding several of these enzymes have been cloned (8-12), as have genes for other amino acid biosynthetic enzymes based on their structural or functional homology to microbial or mammalian enzymes (13-17).Although histidine biosynthesis has been elucidated in several prokaryotic and eukaryotic microbes (18-22), the biosynthetic pathway in higher plants is unknown (23). Indirect evidence has indicated that the pathway follows a route similar to that found in bacteria and fungi (24,25). Recently, histidinol dehydrogenase (HDH; L-histidinol:NAD+ oxidoreductase, EC 1.1.1.23) was purified to homogeneity from Brassica oleracea (cabbage; ref. 26), proving that the final steps in histidine biosynthesis proceed in plants as they do in prokaryotes and fungi.In this report, we describe the isolation ofa full-length cDNA encoding HDH from cabbage. The plant coding sequence ¶ was approximately 50o identical to known microbial genes. To demonstrate function of the plant clone, the cDNA was expressed in an Escherichia coli strain lacking the histidine operon. The cDNA was found to suppress the his deletion when the bacteria were grown in the presence of histidinol. MATERIALS AND METHODSPlant Material and Bacterial Strains. Mature spring cabbage (Brassica oleracea L. var capitata L.) was purchased from a local...

Histidinol dehydrogenase (HDH), a dimeric protein, catalyzes two sequential oxidation reactions to yield L-histidine from L-histidinol via L-histidinal. HDH contains 1 mol of Zn(II) per mol of subunit, and removal of this metal abolishes the enzymatic activity. On substitution of Zn(II) with 113Cd(II), the enzyme ([113Cd]HDH) showed similar catalytic activity. The 113Cd NMR spectra of [113Cd]HDH were measured under various conditions. The 113Cd NMR spectrum of [113Cd]HDH showed a resonance at 110 ppm, which indicates that the metal ion is bound to the protein by a combination of nitrogen and oxygen ligands. 113Cd NMR spectra of [113Cd]HDH were measured as complexes with two substrates (L-histidinol and DL-histidinal) and four inhibitors (imidazole, histamine, L-histidine, and DL-4-(4-imidazolyl)-3-amino-2-butanone) in the absence and presence of NAD+. Significant shifts of [113Cd]-HDH resonance in the presence of the ligand indicate that the metal ion is located in the catalytic site of HDH and that substrates and inhibitors interact with the metal ion. The role of the metal ion in the HDH reaction is discussed.

Histidinol dehydrogenase (HDH), a Zn-metalloenzyme, produces His from histidinol through two successive oxidation reactions with NAD+ as a coenzyme. A mutation, His261-->Asn, caused the complete loss of the Zn, thereby inactivating the enzyme, without significant structural perturbation. The ability to oxidize an intermediate, histidinaldehyde, was restored to about 4% of that of the wild-type enzyme by adding 0.5 mM MnCl2, whereas the histidinol oxidation activity could not be recovered with the mental addition. We concluded that the His residue at position 261 is essential for the ligation of the Zn of cabbage HDH.

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