Crystal Structures of a Hyperthermophilic Archaeal Homoserine Dehydrogenase Suggest a Novel Cofactor Binding Mode for Oxidoreductases

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 NADdependent 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.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] . In this pathway, aspartate is first phosphorylated to β -aspartyl phosphate (β -Ap) by aspartate kinase, after which L-aspartate-β -semialdehyde (Asa) dehydrogenase catalyzes the conversion of β -Ap to Asa. The third enzyme in the pathway, HseDH, catalyzes the NAD(P)H-dependent reduction of Asa to Hse. Subsequent metabolism of Hse yields methionine, threonine or isoleucine, which are all essential in humans. Because HseDH is central to the synthesis of these amino acids, regulation of the enzyme by its end products has been extensively studied in bacteria, yeast and plants at both the activity and genetic levels 4-7 . In particular, threonine-dependent feedback regulation to control HseDH activity has been analyzed in detail. Additionally, HseDH is thought to be a potential target for the structure-based design of antibiotics or herbicides, as the enzyme is not present in mammals 1,2,8 . However, information about the structure of HseDH remains limited. Indeed, the crystal structures of only the Saccharomyces cerevisiae and Thermus thermophilus enzymes have so far been reported 9,10 .

Biochemical Characterization of Homoserine Dehydrogenase from Pseudomonas aeruginosa

“…In addition, we also carried out size‐exclusion chromatography, circular dichroism spectroscopy, and thermal denaturation test. In particular, the size‐exclusion chromatography demonstrated that PaHSD exists as a tetramer in solution, which is in contrast to the previously reported HSDs from other species that commonly form dimers …”

“…Structural studies on HSDs from other microorganisms have revealed that HSD consists of three domains: the nucleotide‐binding domain with a characteristic α/β Rossmann fold, the substrate‐binding domain, and the dimerization domain . Even though structural and enzymatic characterization studies have been reported for various microorganisms, studies on HSD from Pseudomonas aeruginosa , which is a ubiquitous environmental bacterium causing one of the top three opportunistic human infections, have not yet been reported .…”

Unique coenzyme binding mode of hyperthermophilic archaealsn-glycerol-1-phosphate dehydrogenase fromPyrobaculum calidifontis

“…Recently, we determined the crystal structure of a homoserine dehydrogenase (HseDH) from the hyperthermophilic archaeon Pyrococcus horikoshii . Similar to P. calidifontis G1PDH, the refinement of the structure and mass analysis showed the presence of the bound cofactor NADPH, although it had not been added during crystallization.…”

“…In the case of P. horikoshii HseDH, the NADPH molecule is positioned/configured correctly in the nucleotide‐binding site similar to the case of conventional NADP(H)‐dependent dehydrogenases. Therefore, we have proposed that the enzyme exhibits a new variation on a molecular basis for the cofactor preference of dehydrogenase: the very strong binding of NADP acts as an obstacle to NAD(P)‐dependent dehydrogenase catalytic activity . With P. calidifontis G1PDH, in contrast, the enzyme shows an unusual binding for NADPH.…”

Molecular Mechanisms of Enzyme Activation by Monovalent Cations

“…The formyltransferase of the archaeon Methanopyrus kandleri utilizes high concentrations of K ϩ for activity and thermostability (92). Na ϩ binding sites have been reported in archaeal dehydrogenases (93,94) and aldehyde ferredoxin oxidoreductase of the hyperthermophile Pyrococcus furiosus (95). The architecture of these sites has been retained during evolution (6).…”