Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

Abstract

Crystal structures of the unique hexokinase KlHxk1 of the yeast Kluyveromyces lactis were determined using eight independent crystal forms. In five crystal forms, a symmetrical ringshaped homodimer was observed, corresponding to the physiological dimer existing in solution as shown by small-angle x-ray scattering. The dimer has a head-to-tail arrangement such that the small domain of one subunit interacts with the large domain of the other subunit. Dimer formation requires favorable interactions of the 15 N-terminal amino acids that are part of the large domain with amino acids of the small domain of the opposite subunit, respectively. The head-to-tail arrangement involving both domains of the two KlHxk1 subunits is appropriate to explain the reduced activity of the homodimer as compared with the monomeric enzyme and the influence of substrates and products on dimer formation and dissociation. In particular, the structure of the symmetrical KlHxk1 dimer serves to explain why phosphorylation of conserved residue Ser-15 may cause electrostatic repulsions with nearby negatively charged residues of the adjacent subunit, thereby inducing a dissociation of the homologous dimeric hexokinases KlHxk1 and ScHxk2. The enzymes of the hexokinase family catalyze the intracellular trapping and the initiation of metabolism of glucose, fructose, and mannose. In addition to their role in glycolysis, an increasing number of yeast, plant, and mammalian hexokinases have been found to represent multifunctional proteins that are implicated in glucose sensing and signaling (1-4), whereas their glycolytic sugar substrate plays a dual role as a carbon source and hormone-like regulator (4, 5). The molecular basis underlying the involvement of hexokinases in the transcriptional control of glucose metabolism and in glucose homeostasis is their ability to interact with mitochondria and to reversibly translocate to nuclei (3, 6 -9).In the Crabtree-positive yeast Saccharomyces cerevisiae, glucose abundance is accompanied by the translocation of the cytosolic hexokinase isoenzyme 2 (ScHxk2) 4 and the transcriptional repressor Mig1 (ScMig1) into the nucleus, where both proteins participate in the formation of a hetero-oligomeric complex that suppresses the transcription of ScMig1 target genes like SUC2 encoding invertase (9). The mechanism of glucose signaling in glucose-repressible strains of the Crabtree-negative yeast Kluyveromyces lactis, used increasingly as a model organism in comparative functional genomics (10 -12), is largely unknown; however, the unique hexokinase KlHxk1 encoded by the RAG5 gene, the expression of glucose transporters, and the capacity for glucose transport seem to be involved (13)(14)(15).Contrary to the situation in bakers' yeast, glucose and fructose limitation causes the translocation of the mammalian hexokinase isoenzyme IV (also referred to as "glucokinase" or hexokinase D) to the nucleus of the liver parenchymal cell where it binds to its regulatory protein, GKRP, and retranslocates when glucose is abundantly av...

Biochemical and structural study ofArabidopsishexokinase 1

“…Both structures share the same hexokinase fold with a typical palm shape as reported previously (Anderson, McDonald et al, 1978;Anderson, Stenkamp et al, 1978;Bennett & Steitz, 1978;Aleshin, Zeng, Bourenkov et al, 1998;Mulichak et al, 1998;Kuettner et al, 2010;Kamata et al, 2004;Figs. Both structures share the same hexokinase fold with a typical palm shape as reported previously (Anderson, McDonald et al, 1978;Anderson, Stenkamp et al, 1978;Bennett & Steitz, 1978;Aleshin, Zeng, Bourenkov et al, 1998;Mulichak et al, 1998;Kuettner et al, 2010;Kamata et al, 2004;Figs.…”

“…An extensive hydrogen-bonding network was observed between the hydroxyl groups of glucose and the side chains of Thr194, Lys195, Asn229, Asp230, Asn256, Glu284 and Glu315, as well as an ordered water molecule within the cleft (Fig. AtHXK1 thus binds glucose in a mode quite similar to those of HXKs and GKs Aleshin, Zeng, Bourenkov et al, 1998;Kuettner et al, 2010;Lunin et al, 2004;Rosano et al, 1999). The residues of the glucose-binding site are conserved in all known HXKs and GKs except fructokinases (FRKs) ( Supplementary Fig.…”

“…of 1.7 Å over 449 C atoms. Both homologues form complexes with glucose, and they share sequence identities of 34 and 35%, respectively, with liganded AtHXK1 and Glc-AtHXK1 proceeds in the same manner as that in HsHXK-I, KlHXK1 and HsGK, although each one rotates to a different extent (Kamata et al, 2004;Aleshin, Zeng, Bourenkov et al, 1998;Kuettner et al, 2010). The DALI server also showed that Glc-AtHXK1 is homologous to HsGK (PDB entry 4ixc; Waring et al, 2013) and Schistosoma mansoni HXK1 (SmHXK1; PDB entry 1bdg; Mulichak et al, 1998).…”

Plasmodium vivaxand human hexokinases share similar active sites but display distinct quaternary architectures

“…1(b), Supplementary Fig. S3(a)] (Kuettner et al, 2010;Feng et al, 2015;Whittington et al, 2015). In both conformations the density for the small domain is weak, suggesting that this region is more flexible than the large domain [ Fig.…”

HXK1 regulates carbon catabolism, sporulation, fumonisin B1 production and pathogenesis in Fusarium verticillioides

“…EU247513), which was designated previously as HXK1 (Bluhm et al, 2008), was selected for characterization based on its expression profile during kernel colonization (Bluhm et al, 2008) and the similarity of the predicted Hxk1 protein to a functionally characterized hexokinase from Kluyveromyces lactis (KlHxk1, GenBank accession no. CAA08922.1; Kuettner et al, 2010). HXK1 has an ORF of 2329 bp with five introns, and is predicted to encode a protein of 493 aa containing two structurally similar domains.…”