Molecular Structure of Saccharomyces cerevisiae Gal1p, a Bifunctional Galactokinase and Transcriptional Inducer

Abstract

Gal1p of Saccharomyces cerevisiae is capable of performing two independent cellular functions. First, it is a key enzyme in the Leloir pathway for galactose metabolism where it catalyzes the conversion of ␣-D-galactose to galactose 1-phosphate. Second, it has the capacity to induce the transcription of the yeast GAL genes in response to the organism being challenged with galactose as the sole source of carbon. This latter function is normally performed by a highly related protein, Gal3p, but in its absence Gal1p can induce transcription, albeit inefficiently, both in vivo and in vitro. Here we report the x-ray structure of Gal1p in complex with ␣-D-galactose and Mg-adenosine 5-(␤,␥-imido)triphosphate (AMPPNP) determined to 2.4 Å resolution. Overall, the enzyme displays a marked bilobal appearance with the active site being wedged between distinct N-and C-terminal domains. Despite being considerably larger than other galactokinases, Gal1p shares a similar molecular architecture with these enzymes as well as with other members of the GHMP superfamily. The extraordinary levels of similarity between Gal1p and Gal3p (ϳ70% amino acid identity and ϳ90% similarity) have allowed a model for Gal3p to be constructed. By identifying the locations of mutations of Gal3p that result in altered transcriptional properties, we suggest potential models for Gal3p function and mechanisms for its interaction with the transcriptional inhibitor Gal80p. The GAL genetic switch has long been regarded as a paradigm for the control of gene expression in eukaryotes. Understanding the manner in which two of the proteins that function in transcriptional regulation interact with one another is an important step in determining the overall molecular mechanism of this switch.There are four enzymes in the Leloir pathway that are responsible for the conversion of ␤-D-galactose to the more metabolically useful glucose 1-phosphate (1). In the first step of this pathway, ␤-D-galactose is epimerized to ␣-D-galactose by galactose mutarotase. The following step involves the ATP-dependent addition of a phosphate group to the galactose C-1 hydroxyl by galactokinase to yield galactose 1-phosphate. Subsequently, a UMP moiety, derived from UDP-glucose, is transferred to galactose 1-phosphate to generate glucose 1-phosphate and UDPgalactose. This reaction is catalyzed by galactose-1-phosphate uridylyltransferase. To complete the pathway, UDP-galactose is converted to UDP-glucose by UDP-galactose 4-epimerase.

Induction of the gal Pathway and Cellulase Genes Involves No Transcriptional Inducer Function of the Galactokinase in Hypocrea jecorina

“…The presence of ScGal3 seems to be a unique trait of S. cerevisiae, and its role as a transcriptional inducer is fulfilled in other yeasts such as in Kluyveromyces lactis by a single galactokinase KlGal1. The regulatory function of KlGal1 does not require the galactokinase activity (6), which is consistent with the recent demonstration that the enzymatic and the weak transcriptional inducer properties of ScGal1 are located in separate parts of the protein (7).…”

Synergistic dual positive feedback loops established by molecular sequestration generate robust bimodal response

“…Gal1p is a close homolog of Gal3p and has been shown to interact with Gal80p with a weaker affinity than Gal3p (14,15). Furthermore, a GAL3 deletion strain was shown to induce GAL gene expression at a significantly slower rate compared with WT, whereas cells with combined GAL1 and GAL3 deletions fail to activate their GAL pathway (16).…”

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

“…In recent years, the crystal structures of the galactokinases from Lactococcus lactis complexed with galactose (Thoden & Holden, 2003), Pyrococcus furiosus with ADP and galactose (Hartley et al, 2004), human with adenosine 5 0 -( , -imino)-triphosphate (AMP-PNP) and galactose and Saccharomyces cerevisiae with AMP-PNP and galactose (Thoden, Sellick et al, 2005) have been solved to 2.1, 2.9, 2.4 and 2.5 Å resolution, respectively. The structures were all in ligand-bound forms; therefore, determination of both apo and ligand-bound structures may shed light on the catalytic mechanism of galactokinases.…”