Identification and characterization of a novel glucose-phosphorylating enzyme in<i>Kluyveromyces lactis</i>

Kettner, Karina; Müller, Eva‐Christina; Otto, Albrecht; Rödel, Gerhard; Breunig, Karin D.; Kriegel, Thomas

doi:10.1111/j.1567-1364.2007.00259.x

Cited by 11 publications

(15 citation statements)

References 43 publications

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“…The slow growth on glucose (Fig. 1A) is consistent with the expression of a recently identified glucokinase (KlGlk1) of unknown physiological function in strain JA6⌬rag5 (12). Secondly, the rate of phosphorylation of glucose and fructose determined according to Ref.…”

Section: Klhxk1 Deficiency Of Mutant Strain Ja6⌬rag5 Of K Lactis-supporting

confidence: 83%

“…With respect to glucose phosphorylation and signaling, the genome duplication event is associated with the expression of two hexokinases (ScHxk1 and ScHxk2), one glucokinase (ScGlk1), and one glucokinase paralog (ScEmi2). In comparison, the genome of the Crabtreenegative model organism Kluyveromyces lactis, in which no genome duplication has occurred (10), encodes a single hexokinase (KlHxk1) (11) and a single glucokinase (KlGlk1) (12). Prominent physiological features of K. lactis are its growth on lactose as the sole carbon source (13), the limited exploitation of its glucose uptake capacity during aerobic growth, and the low extent of aerobic ethanol accumulation (14).…”

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confidence: 99%

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Proteomic and Functional Consequences of Hexokinase Deficiency in Glucose-repressible Kluyveromyces lactis

Mates

Kettner

Heidenreich

et al. 2014

Molecular & Cellular Proteomics

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The analysis of glucose signaling in the Crabtree-positive eukaryotic model organism Saccharomyces cerevisiae has disclosed a dual role of its hexokinase ScHxk2, which acts as a glycolytic enzyme and key signal transducer adapting central metabolism to glucose availability. In order to identify evolutionarily conserved characteristics of hexokinase structure and function, the cellular response of the Crabtree-negative yeast Kluyveromyces lactis to rag5 null mutation and concomitant deficiency of its unique hexokinase KlHxk1 was analyzed by means of difference gel electrophoresis. In total, 2,851 fluorescent spots containing different protein species were detected in the master gel representing all of the K. lactis proteins that were solubilized from glucose-grown KlHxk1 wildtype and mutant cells. Mass spectrometric peptide analysis identified 45 individual hexokinase-dependent proteins related to carbohydrate, short-chain fatty acid and tricarboxylic acid metabolism as well as to amino acid and protein turnover, but also to general stress response and chromatin remodeling, which occurred as a consequence of KlHxk1 deficiency at a minimum 3-fold enhanced or reduced level in the mutant proteome. In addition, three proteins exhibiting homology to 2-methylcitrate cycle enzymes of S. cerevisiae were detected at increased concentrations, suggesting a stimulation of pyruvate formation from amino acids and/or fatty acids. Experimental validation of the difference gel electrophoresis approach by post-lysis dimethyl labeling largely confirmed the abundance changes detected in the mutant proteome via the former method. Taking into consideration the high proportion of identified hexokinase-dependent proteins exhibiting increased proteomic levels, KlHxk1 is likely to have a repressive function in a multitude of metabolic pathways. The proteomic alterations detected in the mutant classify KlHxk1 as a multifunctional enzyme and support the view of evolutionary conservation of dual-role hexokinases even in organisms that are less specialized than S. cerevisiae in terms of glucose utilization. Molecular & Cellular

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Section: Klhxk1 Deficiency Of Mutant Strain Ja6⌬rag5 Of K Lactis-supporting

confidence: 83%

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confidence: 99%

Proteomic and Functional Consequences of Hexokinase Deficiency in Glucose-repressible Kluyveromyces lactis

Mates

Kettner

Heidenreich

et al. 2014

Molecular & Cellular Proteomics

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“…In contrast, no data are available on the covalent modification and/or intracellular distribution of KlHxk1 in the nonfermentative yeast K. lactis. The two hexokinases exhibit 73% sequence identity (22), and dimerization of the K. lactis enzyme apparently decreases its glucose kinase activity (23), as observed similarly for ScHxk2 (21). In addition, the monomer-dimer equilibrium of both hexokinases depends in a comparable manner on the enzyme concentration and on the presence of substrates and products with glucose most strongly inducing homodimer dissociation (21,23).…”

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confidence: 78%

“…4A). It should be noted, however, that the latter two structures do not necessarily reflect a substrateinduced conformational transition because they belong to different isoenzymes of S. cerevisiae hexokinase (29,30), which share 77% sequence identity (22).…”

Section: Resultsmentioning

confidence: 99%

“…Keeping in mind both the considerable degree of sequence identity between KlHxk1 and ScHxk2 (73%) (22), with a striking conservation of amino acids in the intersubunit interface including the in vivo phosphorylation site, Ser-15, and the comparably high degree of identity (70.6% in an 85-residues overlap) in the N-terminal part of the KlMig1 and ScMig1 proteins, a mechanism of Mig1-hexokinase interaction similar to that proposed for S. cerevisiae may also be anticipated for K. lactis. Indeed, preliminary studies on immobilized KlMig1 13-mer peptides and isolated KlHxk1 enzyme using anti-KlHxk1 antibodies for enzymepeptide complex detection suggest complex formation.…”

Section: Discussionmentioning

confidence: 99%

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Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

Kuettner

Kettner

Keim

et al. 2010

Journal of Biological Chemistry

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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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Current awareness on yeast

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In order to keep subscribers up‐to‐date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly‐published material on yeasts. Each bibliography is divided into 10 sections. 1 Reviews; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (5 weeks journals ‐ search completed 5th. Dec. 2007)

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Identification and characterization of a novel glucose-phosphorylating enzyme inKluyveromyces lactis

Cited by 11 publications

References 43 publications

Proteomic and Functional Consequences of Hexokinase Deficiency in Glucose-repressible Kluyveromyces lactis

Proteomic and Functional Consequences of Hexokinase Deficiency in Glucose-repressible Kluyveromyces lactis

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

Current awareness on yeast

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