Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase from<i>Pyrococcus horikoshii</i>

Inagaki, E.; Sakamoto, Keiko; Obayashi, Naomi; Terada, Takaho; Shirouzu, Mikako; Bessho, Yoshitaka; Kuroishi, Chizu; Kuramitsu, Seiki; Shinkai, Akeo; Yokoyama, Shigeyuki

doi:10.1107/s1744309106001813

Cited by 6 publications

(4 citation statements)

References 13 publications

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“…It is likely, therefore, that ligand binding results in a small, more energetically favorable conformational change that stabilizes the protein and forms or unmasks the galactose-binding site. There is an unpublished structure of an unliganded galactokinase in the Protein Data Bank (accession number 2CZ9) arising from the crystallization of the Pyrococcus horikoshii enzyme (23). This structure appears to be very similar to the liganded versions (11,24,25), which supports the notion that a large conformational change does not occur within the galactokinases upon ligand binding.…”

Section: The Galactokinase Activity Of Scgal1p Was Inhibited By Gal80mentioning

confidence: 66%

The Effect of Ligand Binding on the Galactokinase Activity of Yeast Gal1p and Its Ability to Activate Transcription

Sellick

Jowitt

Reece

2009

Journal of Biological Chemistry

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The galactokinase from Saccharomyces cerevisiae (ScGal1p) is a bifunctional protein. It is an enzyme responsible for the conversion of ␣-D-galactose into galactose 1-phosphate at the expense of ATP but can also function as a transcriptional inducer of the yeast GAL genes. For both of these activities, the protein requires two ligands; a sugar (galactose) and a nucleotide (ATP). Here we investigate the effect of these ligands on the stability and conformation of ScGal1p to determine how the ligands alter protein function. We show that nucleotide binding increases the thermal stability of ScGal1p, whereas binding of galactose alone had no effect on the stability of the protein. This nucleotide stabilization effect is also observed for the related proteins S. cerevisiae Gal3p and Kluyveromyces lactis Gal1p and suggests that nucleotide binding results in the formation of, or the unmasking of, the galactose-binding site. We also show that the increase in stability of ScGal1p does not result from a large conformational change but is instead the result of a smaller more energetically favorable stabilization event. Finally, we have used mutant versions of ScGal1p to show that the galactokinase and transcriptional induction functions of the protein are distinct and separable. Mutations resulting in constitutive induction do not function by mimicking the more stable active conformation but have highlighted a possible site of interaction between ScGal1p and ScGal80p. These data give significant insights into the mechanism of action of both a galactokinase and a transcriptional inducer.The yeasts Saccharomyces cerevisiae and Kluyveromyces lactis are both capable of utilizing galactose as a source of carbon. To do so, they activate the genes encoding the enzymes of the Leloir pathway, collectively termed the GAL genes (1). The transcriptional switch controlling GAL gene expression is composed of an activator (Gal4p), an inhibitor (Gal80p), and a ligand sensor/transcriptional inducer (Gal3p in S. cerevisiae or Gal1p in K. lactis). The GAL regulatory proteins from the two yeasts are at least in part, interchangeable. For example, Gal4p from both S. cerevisiae (ScGal4p) and K. lactis (KlGal4p) will complement a gal4 mutation in either yeast (2, 3) despite the two proteins sharing comparatively little overall sequence similarity (28% amino acid identity and 57% similarity over their entire length). Gal80p from either yeast are highly related (58% amino acid identity and 82% similarity) and will inhibit the transcriptional activity of either version of Gal4p (4). However, although KlGal1p can complement both a Scgal1 (galactokinase-defective) and Scgal3 (ligand sensor-defective) mutation (5), ScGal3p cannot complement the non-inducible phenotype of a Klgal1 deletion mutant unless the KlGAL80 gene is also replaced by ScGAL80 (6).ScGal1p and ScGal3p are extraordinarily similar proteins (ϳ70% amino acid identity and ϳ90% similarity). Both proteins require galactose and ATP to function. ScGal1p utilizes the ligands as a galactokinase...

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Section: The Galactokinase Activity Of Scgal1p Was Inhibited By Gal80mentioning

confidence: 66%

The Effect of Ligand Binding on the Galactokinase Activity of Yeast Gal1p and Its Ability to Activate Transcription

Sellick

Jowitt

Reece

2009

Journal of Biological Chemistry

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“…The aforementioned three enzymes comprising the Leloir pathway are widely distributed in bacteria and eukarya, and structural and functional analyses of several examples have been reported (Holden et al, 2003). Galactokinases from the hyperthermophilic archaea Pyrococcus furiosus and Pyrococcus horikoshii (Verhees et al, 2002;Inagaki et al, 2006;Hartley et al, 2004) have also been characterized and the molecular basis of substrate recognition by the P. furiosus enzyme is now known. However, information about the other two enzymes involved in the Leloir pathway in archaea remains limited.…”

Section: Introductionmentioning

confidence: 99%

Expression, purification, crystallization and preliminary X-ray diffraction analysis of a galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeonPyrobaculum aerophilum

et al. 2012

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A galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 8000 as the precipitant. The crystals belonged to the tetragonal space group P4 1 , with unit-cell parameters a = b = 73.3, c = 126.1 Å , and diffracted to 2.73 Å resolution on beamline BL5A at the Photon Factory. The overall R merge was 7.3% and the data completeness was 99.8%.

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“…However, because the structure of any galactokinase has not been solved in the absence of ligands (5,18,23,24), it is difficult to speculate as to the state of the apoprotein. Recently, however, crystals of the galactokinase from Pyrococcus horikoshii in both the apo-and ligand-bound forms have been reported (25).…”

Section: Discussionmentioning

confidence: 99%

Contribution of Amino Acid Side Chains to Sugar Binding Specificity in a Galactokinase, Gal1p, and a Transcriptional Inducer, Gal3p

Sellick

Reece

2006

Journal of Biological Chemistry

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The crystal structure of the yeast galactokinase, Gal1p, in the presence of its substrates has been solved recently. We systematically mutated each of the amino acid side chains that, from the structure, are implicated to be involved in direct contact with the hydroxyl groups of the galactose ring. One of these mutations, D62A, abolished all detectable galactokinase activity but retained the ability to use D-glucose as a substrate. Mutation of Asp-62 to either leucine, phenylalanine, or histidine resulted in the formation of protein with similar characteristics to D62A. Yeast galactokinase is highly similar to Gal3p, the ligand sensor and transcriptional inducer of the GAL genes. Equivalent mutations in Gal3p also abolished its ability to respond to galactose and uncovered its ability to respond to D-glucose. It therefore appears that Gal1p and Gal3p respond to their substrates in a similar, perhaps identical, fashion. This work also validates the approach of screening for mutants in an easily assayable system prior to mutant analysis in a more experimentally difficult transcriptional regulator.The yeast Saccharomyces cerevisiae contains within its genome two galactokinase-like genes. The first of these, GAL1, encodes Gal1p, a bona fide galactokinase whose major role in the cell is to convert ␣-Dgalactose into galactose 1-phosphate at the expense of ATP as part of the Leloir pathway, which is responsible for the conversion of ␤-Dgalactose to the more metabolically useful glucose 6-phosphate (1). The second galactokinase-like gene, GAL3, encodes Gal3p, which has no detectable galactokinase activity itself but, rather, interacts with galactose and ATP to regulate the transcription of the Leloir pathway enzyme genes (2). Gal1p and Gal3p are highly similar proteins, sharing some 70% identity and 90% similarity at the amino acid level. This similarity has been underscored by the observation that the insertion of just two amino acids into Gal3p (a serine and an alanine inserted directly after amino acid 164) imparts the resulting protein with galactokinase activity (3).The kinetic mechanism of Gal1p has been described as proceeding via an ordered, ternary complex in which ATP binds first and galactose second (4). The most likely explanation for an ordered mechanism is that the binding of the first substrate induces a conformational change in the enzyme resulting in the formation of a high affinity binding site for the second substrate. The structure of Gal1p, complexed with galactose and a nonhydrolyzable ATP analogue, has been solved recently (5). The enzyme adopts a bilobal appearance with the active site being wedged between distinct amino-and carboxyl-terminal domains. The sugar is held in the active site by a network of potential hydrogen bond interactions (Fig. 1). This network, composed of residues Arg-53, , is capable of contacting each of the five hydroxyl groups of the sugar ring. To explore the requirement of this hydrogen bond network for interaction with the sugar, we undertook a mutational analysis of th...

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Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase fromPyrococcus horikoshii

Cited by 6 publications

References 13 publications

The Effect of Ligand Binding on the Galactokinase Activity of Yeast Gal1p and Its Ability to Activate Transcription

The Effect of Ligand Binding on the Galactokinase Activity of Yeast Gal1p and Its Ability to Activate Transcription

Expression, purification, crystallization and preliminary X-ray diffraction analysis of a galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeonPyrobaculum aerophilum

Contribution of Amino Acid Side Chains to Sugar Binding Specificity in a Galactokinase, Gal1p, and a Transcriptional Inducer, Gal3p

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