Abstract
NAD(P)-dependent dehydrogenases differ according to their coenzyme preference: some prefer NAD, others NADP, and still others exhibit dual cofactor specificity. The structure of a newly identified archaeal homoserine dehydrogenase showed this enzyme to have a strong preference for NADP. However, NADP did not act as a cofactor with this enzyme, but as a strong inhibitor of NAD-dependent homoserine oxidation. Structural analysis and site-directed mutagenesis showed that the large number of interactions between the cofactor and the enzyme are responsible for the lack of reactivity of the enzyme towards NADP. This observation suggests this enzyme exhibits a new variation on cofactor binding to a dehydrogenase: very strong NADP binding that acts as an obstacle to NAD(P)-dependent dehydrogenase catalytic activity.
Highlights
Homoserine dehydrogenase (HseDH, EC 1.1.1.3) is a key enzyme in the biosynthetic pathway from aspartate to homoserine (Hse), which is a common precursor for the synthesis of three amino acids, methionine, threonine and isoleucine in plants and microorganisms[1,2,3]
SDS-PAGE showed the subunit molecular mass of P. horikoshii HseDH to be about 40 kDa, which is consistent with the molecular weight (36,925) calculated from the amino acid sequence
Even after incubation for 10 min at 95 °C, the enzyme retained 70% of its activity, making P. horikoshii HseDH the most thermostable HseDH described to date
Summary
Molecular and catalytic properties of P. horikoshii HseDH. After transforming Escherichia coli with pCHseDH, an expression vector encoding His-tagged HseDH, the crude extract from the recombinant cells exhibited strong HseDH activity, and the enzyme was readily purified in two simple steps: heat treatment and affinity column chromatography. SDS-PAGE showed the subunit molecular mass of P. horikoshii HseDH to be about 40 kDa, which is consistent with the molecular weight (36,925) calculated from the amino acid sequence. In the S. cerevisiae enzyme, the coordination sphere surrounding the ion consists of the backbone carbonyl atoms of Glu[143], Val[146], Ala[148] and Leu[150], and a side-chain oxygen of Glu[143] Of these residues, Glu[143], Val[146] and Ala[148] are conserved as Glu[140], Val[143] and Ala[145], respectively, in P. horikoshii HseDH, but Leu[150] is replaced by Thr[147]. The sequence alignment showed that 22 residues in the corresponding region in S. cerevisiae HseDH are not present in P. horikoshii HseDH (Fig. 2). Another noteworthy difference was in the structure of the chain located at the bottom of the nucleotide-binding domain.
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