Salmonella typhimurium histidinol dehydrogenase: complete reaction stereochemistry and active site mapping

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Abstract
The stereochemistry of the L-histidinol dehydrogenase reaction was determined to be R and NAD for both steps, confirming previous results with a fungal extract [Davies, D., Teixeira, A., and Kenworthy, O. (1972) Biochemm. J. 127, 335-343]. NMR analysis of monodeuteriohistidinols produced by histidinol/NADH exchange reactions arising via reversal of the alcohol oxidation reaction indicated a single stereochemistry at histidinol for that step. Comparison of vicinal coupling values of the exchange products with those of L-alaninol and a series of (S)-2-amino-1-alcohols allowed identification of the absolute stereochemistry of monodeuteriohistidinols and showed that histidinol dehydrogenase removes first the pro-S then the pro-R hydrogens of substrate histidinol. The enzyme stereochemistry was confirmed by isotope effects of monodeuteriohistidinols as substrates for the pro-R-specific dehydrogenation catalyzed by liver alcohol dehydrogenase. Active site mapping was undertaken to investigate substrate-protein interactions elsewhere in the histidinol binding site. Critical binding regions are the side-chain amino group and the imidazole ring, whose methylation at the 1- or 2-position caused severe decreases in binding affinity. Use of alternative substrates further clarified active site interactions with the substrate. Compounds in which the .alpha.-amino group was replaced by chloro, bromo, or hydrogen substituents were not substrates of the overall reaction at 1/10000 the normal rate. The .alpha.-hydroxy analogue of histidinol was a substrate, with kcat/Km = 0.1% that of histidinol. Removal or alteration of the imidazole ring also prevented catalysis, resulting in complete inactivity with alaninol or thienylalaninol.