not related to vaspin-mediated changes in insulin sensitivity as determined by euglycemic-hyperinsulinemic clamp studies. Improved glucose metabolism could be mediated by increased insulin plasma concentrations 150 min after a glucose challenge in db/db mice, supporting the hypothesis that vaspin may inhibit insulin degradation by hK7 in the circulation. In conclusion, we demonstrate the inhibitory serpin nature and the first protease target of the adipose tissue-derived serpin vaspin, and our findings suggest hK7 inhibition by vaspin as an underlying physiological mechanism for its compensatory actions on obesity-induced insulin resistance.

Despite the fact that many cultures around the world value and utilize garlic as a fundamental component of their cuisine as well as of their medicine cabinets, relatively little is known about the plant's protein configuration that is responsible for the specific properties of garlic. Here, we report the three-dimensional structure of the garlic enzyme alliinase at 1.5 Å resolution. Alliinase constitutes the major protein component in garlic bulbs, and it is able to cleave carbon-sulfur bonds. The active enzyme is a pyridoxal-5-phosphate-dependent homodimeric glycoprotein and belongs to the class I family of pyridoxal-5-phosphate-dependent enzymes. In addition, it contains a novel epidermal growth factorlike domain that makes it unique among all pyridoxal-5-phosphate-dependent enzymes.

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...

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Eukaryotic ATP-dependent 6-phosphofructokinases (Pfks) differ from their bacterial counterparts in a much more complex structural organization and allosteric regulation. Pichia pastoris Pfk (PpPfk) is, with ∼ 1 MDa, the most complex and probably largest eukaryotic Pfk. We have determined the crystal structure of full-length PpPfk to 3.05 Å resolution in the T state. PpPfk forms a (αβγ)(4) dodecamer of D(2) symmetry with dimensions of 161 × 157 × 233 Å mainly via interactions of the α chains. The N-terminal domains of the α and β chains have folds that are distantly related to glyoxalase I, but the active sites are no longer functional. Interestingly, these domains located at the 2 distal ends of this protein along the long 2-fold axis form a (αβ)(2) dimer as does the core Pfk domains; however, the domains are swapped across the tetramerization interface. In PpPfk, the unique γ subunit participates in oligomerization of the αβ chains. This modulator protein was acquired from an ancient S-adenosylmethionine-dependent methyltransferase. The identification of novel ATP binding sites, which do not correspond to the bacterial catalytic or effector binding sites, point to marked structural and functional differences between bacterial and eukaryotic Pfks.