¹¹³Cd Nuclear Magnetic Resonance Studies of Cabbage Histidinol Dehydrogenase

Abstract

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.

Mechanism of action and NAD + -binding mode revealed by the crystal structure of l -histidinol dehydrogenase

“…The presence of His-262 and His-419 in the Zn 2ϩ coordination environment is consistent with previous mutagenesis experiments (15). NMR experiments (16) suggested that the amino group of the ligand (including L-histidinol, histamine, and L-histidine) is not involved in Zn . The best-defined electron density corresponds to the phosphate groups and to the adenosine moiety, whereas the density for the nicotinamide is poorly defined (Fig.…”

“…The Zn 2ϩ ion is essential for enzymatic activity, although other metals such as Mn in forming an active enzyme (13). Biochemical and mutagenesis studies on HisD from S. typhimurium (12,14) and Brassica oleracea (15) have identified two histidine residues, corresponding to His-262 and His-419 from E. coli, as part of the active site, although the precise role of His-262 has not been established (14)(15)(16). NMR measurements indicated that the substrate participates in Zn 2ϩ coordination through its imidazole ND1 and alcohol oxygen atoms (16), and Zn 2ϩ has been proposed to participate directly in catalysis (14,16).…”

Effect of excess cadmium ion on the metal binding site of cabbage histidinol dehydrogenase studied by 113Cd‐NMR spectroscopy

“…Hereafter, for convenience, the corresponding metal binding sites at 110, 83, 22, and −2 ppm will be represented as A, A′, A″, and B, respectively. Thus, in the presence of excess Cd 2+ , Cd 2+ ions at site A readily exchange with free Cd 2+ ions faster than those for sites A′ and A″ although, in the absence of excess 113 Cd 2+ ion, extensive dialysis by Tris‐SO 4 buffer cannot remove 113 Cd 2+ ion from [ 113 Cd]HDH [3]. This fact also suggests that the environmental change around site A (peak at 110 ppm in Fig.…”

“…113 Cd metal (95 atom %) was obtained from ISOTEC Inc. (Miamisburg, OH). [ 113 Cd]SO 4 was prepared as described previously [3]. NAD + and l ‐histidinol were purchased from Sigma (St. Louis, MO).…”

“…HDH used in this study was purified from a nuclear polyhedrosis virus genome expression system containing the plasmid pVL1393 [14, 15]. The purification protocol of [Zn]HDH and the preparation and reconstitution of the apoenzyme are the same as that previously described [3, 16, 17]. Zn analysis of the apoenzyme showed no detectable amounts of Zn ion [17], and reconstituted [ 113 Cd]HDH exhibited the same catalytic activity as [Zn]HDH [3].…”