Mechanism of Control of Arabidopsis thaliana Aspartate Kinase-Homoserine Dehydrogenase by Threonine

Abstract

The regulatory domain of the bifunctional threonine-sensitive aspartate kinase homoserine dehydrogenase contains two homologous subdomains defined by a common loop-alpha helix-loop-beta strand-loop-beta strand motif. This motif is homologous with that found in the two subdomains of the biosynthetic threonine-deaminase regulatory domain. Comparisons of the primary and secondary structures of the two enzymes allowed us to predict the location and identity of the amino acid residues potentially involved in two threonine-binding sites of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase. These amino acids were then mutated and activity measurements were carried out to test this hypothesis. Steady-state kinetic experiments on the wild-type and mutant enzymes demonstrated that each regulatory domain of the monomers of aspartate kinase-homoserine dehydrogenase possesses two nonequivalent threonine-binding sites constituted in part by Gln(443) and Gln(524). Our results also demonstrated that threonine interaction with Gln(443) leads to inhibition of aspartate kinase activity and facilitates the binding of a second threonine on Gln(524). Interaction of this second threonine with Gln(524) leads to inhibition of homoserine dehydrogenase activity.

A Novel Organization of ACT Domains in Allosteric Enzymes Revealed by the Crystal Structure of Arabidopsis Aspartate Kinase

“…Furthermore, the two SAM binding sites are not located at the interface between two ACT domains but are sandwiched between specific loops of ACT1 ( Figure 3B). Finally, ACT2 does not interact with an effector as suggested for TD (Wessel et al, 2000) and AK-HSDH (Paris et al, 2003).…”

A new mutation in the yeast aspartate kinase induces threonine accumulation in a temperature‐regulated way

“…The ACT domain has been found in enzymes that are regulated by amino acids or purines and is involved in regulation by these effectors (Chipman and Shaanan, 2001). In Figure 5, only aspartate kinases for which single mutations affecting enzymatic activity or regulation have been described (Arévalo-Rodríguez et al, 1999;Bareich and Wright, 2003;Farfán, 1997;Follettie et al, 1993;Hernando-Rico et al, 2001;Kalinowski et al, 1991;Kikuchi et al, 1999;Marco-Marin et al, 2003;Martín-Rendón et al, 1993;Ogawa-Miyata et al, 2001;Ohnishi et al, 2002;Omori et al, 1993;Paris et al, 2003) are shown. Most but not all of those mutations affecting regulation are located in the ACT domains.…”

“…A few of them are in the amino acid kinase domain, viz. E282D and E279A in S. cerevisiae (this work; Bareich and Wright, 2003) (12) I522A (12) Q524 (12) AtAK-HD S301Y (8) G345D (8) AlAK E164K (6) K8R (5) E119D (5) R198K (5) D202E (5) E250K (7) G323D (7,6) M318I (6) L325F (6) T352I (7,6) V347M (6) T344M (7) S345L (7,6) EcAKIII S301Y (9) T311Y ( Bareich and Wright, 2003;(2) Arévalo-Rodríguez et al, 1999;(3) Martín-Rendón et al, 1993;(4) Farfán, 1997;(5) Marco-Marin et al, 2003;(6) Ogawa-Miyata et al, 2001;(7) Kikuchi et al, 1999;(8) Hernando-Rico et al, 2001;(9) Kalinowski et al, 1991;(10) Ohnishi et al, 2002;(11) Follettie et al, 1993;(12) Paris et al, 2003;(13) Omori et al, 1993Omori et al, et al, 1999Marco-Marin et al, 2003;O...…”

“…Not surprisingly, the rest of the mutations that affect regulation of the enzyme are located in one of the ACT domains (see Figure 5). Some residues in the ACT domain have recently been described as essential in the interaction with the effector, threonine, in A. thaliana (Paris et al, 2003), viz. I441, Q443 in ACT1 of AK-HDH; and I522, Q524 in ACT2.…”

Analysis of Loss-of-Function Mutants in Aspartate Kinase and Homoserine Dehydrogenase Genes Points to Complexity in the Regulation of Aspartate-Derived Amino Acid Contents

“…S1; Chipman and Shaanan, 2001;Jander and Joshi, 2009). The activity of AK-HSDHs is feedback inhibited by Thr (Muehlbauer et al, 1994;Paris et al, 2003;Rognes et al, 2003;Curien et al, 2005). The inhibition is mediated through the two Thr-binding ACT domains in the middle of the proteins (Supplemental Fig.…”