Novel mutations in 13 probands with galactokinase deficiencyV. Kolosha and E. Anoia contributed equally to this work.

Kolosha, Vladimir O.; Anoia, E.; Céspedes, Carlos de; Gitzelmann, R; Shih, Lisa B.; Casco, T.; Saborío, María Carolina Fernández; Trejos, Rafael; Buist, Neil R. M.; Tedesco, Thomas A.; Skach, William R.; Mitelmann, O.; Ledee, Dolena R.; Huang, Kristen M.; Stambolian, Dwight

doi:10.1002/(sici)1098-1004(200005)15:5<447::aid-humu6>3.3.co;2-d

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“…Furthermore, the extended, glycine‐rich loop linking the first and second strands forms part of the active site and contacts the monosaccharide substrate. Two mutations associated with type II galactosemia (G346S and G349S) are located in this loop 21…”

Section: Resultsmentioning

confidence: 99%

Increased Promiscuity of Human Galactokinase Following Alteration of a Single Amino Acid Residue Distant from the Active Site

Kristiansson¹,

Timson²

2011

ChemBioChem

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Galactokinase catalyses the site- and stereospecific phosphorylation of galactose at the expense of ATP. The specificity of bacterial galactokinase enzymes can be broadened by alteration of a tyrosine residue to a histidine. The effects of altering the equivalent residue in human galactokinase (Tyr379) were investigated by testing all 19 possible variants. All of these alterations, except Y379P, resulted in soluble protein on expression in Escherichia coli and all the soluble variants could catalyse the phosphorylation of galactose, except Y379A and Y379E. The variants Y379C, Y379K, Y379R, Y379S and Y379W were all able to catalyse the phosphorylation of a variety of monosaccharides, including ones that are not acted on by the wild-type enzyme. Novel substrates for these variant galactokinases included D-mannose and D-fructose. The latter monosaccharide is presumed to react in the pyranose configuration. Molecular modelling suggested that the alterations do not cause changes to the overall structure of the enzyme. However, alteration of Tyr379 increases the flexibility of the peptide backbone in regions surrounding the active site. Therefore, it is proposed that alteration of Tyr379 affects the substrate specificity by the propagation of changes in flexibility to the active site, permitting a broader range of compounds to be accommodated.

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Section: Resultsmentioning

confidence: 99%

Increased Promiscuity of Human Galactokinase Following Alteration of a Single Amino Acid Residue Distant from the Active Site

Kristiansson¹,

Timson²

2011

ChemBioChem

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Functional analysis of disease‐causing mutations in human galactokinase

Timson

Reece

2003

European Journal of Biochemistry

101

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Galactokinase (EC 2.7.1.6) catalyzes the first committed step in the catabolism of galactose. The sugar is phosphorylated at position 1 at the expense of ATP. Lack of fully functional galactokinase is one cause of the inherited disease galactosemia, the main clinical manifestation of which is early onset cataracts. Human galactokinase (GALK1) was expressed in and purified from Escherichia coli. The recombinant enzyme was both soluble and active. Product inhibition studies showed that the most likely kinetic mechanism of the enzyme was an ordered ternary complex one in which ATP is the first substrate to bind. The lack of a solvent kinetic isotope effect suggests that proton transfer is unlikely to be involved in the rate determining step of catalysis. Ten mutations that are known to cause galactosemia were constructed and expressed in E. coli. Of these, five (P28T, V32M, G36R, T288M and A384P) were insoluble following induction and could not be studied further. Four of the remainder (H44Y, R68C, G346S and G349S) were all less active than the wild-type enzyme. One mutant (A198V) had kinetic properties that were essentially wild-type. These results are discussed both in terms of galactokinase structure-function relationships and how these functional changes may relate to the causes of galactosemia.Keywords: galactosemia; cataracts; GHMP family kinase; GALK1.Galactose is metabolized by the enzymes of the Leloir pathway [1]. The sugar is first phosphorylated at position 1, then converted to UDP-galactose and glucose-1-phosphate (which can enter the glycolytic pathway) by reaction with UDP-glucose. Defects in the enzymes of the Leloir pathway can result in galactosemia in humans [2,3]. The main symptom of this disease is early onset cataracts although mental retardation is also seen in some patients. In the absence of a functional Leloir pathway, galactose accumulates in the lens of the eye where the enzyme aldose reductase catalyzes its conversion to galactitol [4]. High levels of this compound in lens fibre cells cause the uptake of water by osmosis, swelling of the cells, cells lysis and ultimately cataracts. The condition is treated by removal of galactose and lactose from the diet.Galactokinase belongs to a family of small molecule kinases, the GHMP (galactokinase, homoserine kinase, mevalonate kinase, phosphomevalonate kinase) family as defined by sequence similarity [5]. Although there has been no three-dimensional structure of a galactokinase reported to date, structures of homoserine kinase [6,7], mevalonate kinase [8,9] and phosphomevalonate kinase [10] have been completed along with another family member mevalonate-5-diphosphate decarboxylase [11]. Five highly conserved motifs have been identified in galactokinases from different species [12]. The structures of GHMP kinases show a high degree of overall similarity. From this, functions can be inferred for some of the conserved motifs in galactokinase. Motif III is well conserved throughout the GHMP family and interacts with the phosphates of ATP. Motif V, ...

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Novel mutations in 13 probands with galactokinase deficiencyV. Kolosha and E. Anoia contributed equally to this work.

Cited by 2 publications

References 0 publications

Increased Promiscuity of Human Galactokinase Following Alteration of a Single Amino Acid Residue Distant from the Active Site

Increased Promiscuity of Human Galactokinase Following Alteration of a Single Amino Acid Residue Distant from the Active Site

Functional analysis of disease‐causing mutations in human galactokinase

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