Cloning of a human galactokinase gene (GK2) on chromosome 15 by complementation in yeast.

Lee, Robert T.; Peterson, Craig L.; Calman, Andrew F.; Herskowitz, Ira; O’Donnell, James J.

doi:10.1073/pnas.89.22.10887

Cited by 29 publications

(8 citation statements)

References 37 publications

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“…A second gene encoding a putative galactokinase, with ϳ35% amino acid sequence identity, was subsequently reported that mapped to chromosome 15, suggesting, perhaps, that humans contain two galactokinases (12). These apparently contradicting reports for the chromosomal location of human galactokinase have been reconciled through the recent studies of Pastuszak et al (13,14), who demonstrated that the gene located on chromosome 15 does not, in fact, encode a bona fide galactokinase but rather a GalNAc kinase (Scheme 1).…”

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confidence: 68%

The Molecular Architecture of Human N-Acetylgalactosamine Kinase

Thoden

Holden

2005

Journal of Biological Chemistry

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Galactokinase plays a key role in normal galactose metabolism by catalyzing the conversion of ␣-D-galactose to galactose 1-phosphate. Within recent years, the three-dimensional structures of human galactokinase and two bacterial forms of the enzyme have been determined. Originally, the gene encoding galactokinase in humans was mapped to chromosome 17. An additional gene, encoding a protein with sequence similarity to galactokinase, was subsequently mapped to chromosome 15. Recent reports have shown that this second gene (GALK2) encodes an enzyme with greater activity against GalNAc than galactose. This enzyme, GalNAc kinase, has been implicated in a salvage pathway for the reutilization of free GalNAc derived from the degradation of complex carbohydrates. Here we report the first structural analysis of a GalNAc kinase. The structure of the human enzyme was solved in the presence of MnAMPPNP and GalNAc or MgATP and GalNAc (which resulted in bound products in the active site). The enzyme displays a distinctly bilobal appearance with its active site wedged between the two domains. The N-terminal region is dominated by a seven-stranded mixed ␤-sheet, whereas the C-terminal motif contains two layers of anti-parallel ␤-sheet. The overall topology displayed by GalNAc kinase places it into the GHMP superfamily of enzymes, which generally function as small molecule kinases. From this investigation, the geometry of the GalNAc kinase active site before and after catalysis has been revealed, and the determinants of substrate specificity have been defined on a molecular level.

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confidence: 68%

The Molecular Architecture of Human N-Acetylgalactosamine Kinase

Thoden

Holden

2005

Journal of Biological Chemistry

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“…described by Kraus et al (28) (31). Another yeast-based expression system has been described for galactokinase, which may be useful in analyzing mutations that cause cataracts in newborns (23). Given and yeast, it is likely that yeast expression assays can be developed for a wide variety of genetic metabolic disorders.…”

Section: Discussionmentioning

confidence: 99%

A yeast system for expression of human cystathionine beta-synthase: structural and functional conservation of the human and yeast genes.

Kruger

Cox

1994

Proc. Natl. Acad. Sci. U.S.A.

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Cystathionine P-synthase (CBS) deficiency in humans is a rare autosomal recessive disease, which is the most common cause of homocystinuria. Individuals with this disorder have a variety of clinical phenotypes including ocular, skeletal, neurological, and cardiovascular defects, which are believed to result primarily from elevated plasma homocysteine levels. Age of onset, symptoms, and severity of the disease can vary greatly among patients (1). Some, but not all, individuals with homocystinuria respond to increased dietary levels of vitamin B6 with an increase in residual enzyme activity associated with a lowering of plasma homocysteine levels. Within sibships, almost complete concordance is observed for responsiveness and nonresponsiveness to vitamin B6 (2). Concordance within sibships is also observed in the residual CBS activity found in cultured fibroblast cells derived from affected individuals (3). Residual enzyme activity in fibroblast extracts ranges from undetectable to 10%6 of normal controls. Generally, vitamin B6 responders have higher levels of residual CBS activity than nonresponders (3-6). These observations suggest that different mutations within the CBS gene alter the enzyme activity in discrete ways; thus, different CBS mutations may be related to the clinically observed heterogeneity found in patients with CBS deficiency.A life-threatening complication of CBS deficiency is thromboembolism (1). Untreated vitamin B6 responsive and nonresponsive individuals have a 4% risk per year of a thrombolytic event (2). Thrombosis can occur at any age and can involve both large and small arteries. Studies also suggest that individuals heterozygous for mutations in CBS are at increased risk for premature peripheral and cerebral arterial disease (7-10). These studies show a correlation between reduced levels of CBS activity and premature vascular disease. Since ='1 in 200 people in the general population is a heterozygote (1), it has been suggested that these individuals may account for a large fraction of those at risk for stroke. However, testing this hypothesis is difficult due to the lack of an accurate assay that identifies heterozygotes (11).To identify and study mutations that cause homocystinuria, an effective assay for the human CBS gene product is required. Such an assay system must be able to distinguish DNA polymorphisms with no phenotypic effect from mutations that affect enzymatic function. In addition, the assay must be able to isolate individual alleles for study.We report here the development of such an experimental system for the study of human CBS using Saccharomyces cerevisiae. We (MATa ura3-52 cys4-1 cys2-1) and WC44a (MATa ura3-52) were constructed by crossing JW1-2c (MATa cys2-1 cys4-1)

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“…Cloning and sequencing of the gene was complicated by the unexpected existence of a second galactokinase-like sequence in the human genome, GALK2. This gene encodes the structurally and functionally related protein N-acetylgalactosamine kinase (EC 2.7.1.157), an enzyme which has only minimal activity towards galactose (Lee et al , 1992;Ai et al , 1995;Agnew & Timson , 2010). The coding sequence for GALK2 was determined three years before that the GALK1 was elucidated in 1995 (Stambolian et al , 1995).…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

The molecular basis of galactosemia — Past, present and future

Timson

2016

Gene

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Galactosemia, an inborn error of galactose metabolism, was first described in the 1900s by von Ruess. The subsequent 100 years have seen considerable progress in understanding the underlying genetics and biochemistry of this condition. Initial studies concentrated on increasing the understanding of the clinical manifestations of the disease. However, Leloir's discovery of the pathway of galactose catabolism in the 1940s and 1950s enabled other scientists, notably Kalckar, to link the disease to a specific enzymatic step in the pathway. Kalckar's work established that defects in galactose 1-phosphate uridylyltransferase (GALT) were responsible for the majority of cases of galactosemia. However, over the next three decades it became clear that there were two other forms of galactosemia: type II resulting from deficiencies in galactokinase (GALK1) and type III where the affected enzyme is UDP-galactose 4'-epimerase (GALE). From the 1970s, molecular biology approaches were applied to galactosemia. The chromosomal locations and DNA sequences of the three genes were determined. These studies enabled modern biochemical studies.Structures of the proteins have been determined and biochemical studies have shown that enzymatic impairment often results from misfolding and consequent protein instability. Cellular and model organism studies have demonstrated that reduced GALT or GALE activity results in increased oxidative stress. Thus, after a century of progress, it is possible to conceive of improved therapies including drugs to manipulate the pathway to reduce potentially toxic intermediates, antioxidants to reduce the oxidative stress of cells or use of "pharmacological chaperones" to stabilise the affected proteins.

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Cloning of a human galactokinase gene (GK2) on chromosome 15 by complementation in yeast.

Cited by 29 publications

References 37 publications

The Molecular Architecture of Human N-Acetylgalactosamine Kinase

The Molecular Architecture of Human N-Acetylgalactosamine Kinase

A yeast system for expression of human cystathionine beta-synthase: structural and functional conservation of the human and yeast genes.

The molecular basis of galactosemia — Past, present and future

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