A molecular approach to galactosemia

Elsas, Louis J.; Langley, Sharon D.; Paulk, E. M.; Hjelm, Lawrence N.; Dembure, Philip P.

doi:10.1007/bf02143798

Cited by 62 publications

(51 citation statements)

References 19 publications

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“…Additionally it was shown that the side chain of Gln-168 provided important hydrogen bonds to both O2 and O5Ј of the nucleotide. 5 It should be noted that in the human enzyme, the mutation of this glutamine to an arginine is the predominant cause of galactosemia among the Caucasian population (42). This change to an arginine residue may result in overstabilization of the enzyme intermediate, thereby compromising its subsequent reaction with galactose 1-phosphate.…”

Section: Minireview: Enzymes Of the Leloir Pathway 43886mentioning

confidence: 99%

Structure and Function of Enzymes of the Leloir Pathway for Galactose Metabolism

Holden

Rayment

Thoden

2003

Journal of Biological Chemistry

441

335

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In most organisms, the conversion of ␤-D-galactose to the more metabolically useful glucose 1-phosphate is accomplished by the action of four enzymes that constitute the Leloir pathway (Scheme 1). In the first step of this pathway, ␤-D-galactose is epimerized to ␣-D-galactose by galactose mutarotase. The next step involves the ATP-dependent phosphorylation of ␣-D-galactose by galactokinase to yield galactose 1-phosphate. As indicated in Scheme 1, the third enzyme in the pathway, galactose-1-phosphate uridylyltransferase, catalyzes the transfer of a UMP group from UDP-glucose to galactose 1-phosphate, thereby generating glucose 1-phosphate and UDP-galactose. To complete the pathway, UDP-galactose is converted to UDP-glucose by UDP-galactose 4-epimerase. In humans, defects in the genes encoding for galactokinase, uridylyltransferase, or epimerase can give rise to the diseased state referred to collectively as galactosemia (1, 2). Although galactosemia is rare, it is potentially lethal with clinical manifestations including intellectual retardation, liver dysfunction, and cataract formation, among others. Indeed, the enzymes of the Leloir pathway have attracted significant research attention for well over 30 -40 years, in part because of their important metabolic role in normal galactose metabolism.As of this year, the three-dimensional structures of all of the enzymes of the Leloir pathway have now been defined. It is thus timely to present in this minireview recent advances in our understanding of the structure and function of these enzymes. For a discussion of the literature prior to 1996, see Ref. 3. Galactose MutarotaseGalactose mutarotase activity was first reported in Escherichia coli in 1965 (4), and the gene encoding it was defined in 1994 (5). Since 1986, genes encoding for proteins with mutarotase activities have been identified in other organisms including Lactococcus lactis (6). With respect to the catalytic mechanism of galactose mutarotase, it was first suggested by Hucho and Wallenfels (7) that the reaction proceeds through the abstraction of the proton from the 1-hydroxyl group of the sugar by an active site base and donation of a proton to the C-5 ring oxygen by an active site acid, thereby leading to ring opening. Subsequent rotation of 180 o about the C-1-C-2 bond followed by abstraction of the proton on the C-5 oxygen and donation of a proton back to the C-1 oxygen generates product. A kinetic analysis of the enzyme from E. coli was recently reported (8).In 2002, the first structure of a galactose mutarotase (from L. lactis) was determined by Thoden et al. (9,10). A ribbon representation of the dimeric enzyme is displayed in Fig. 1. Each subunit contains 339 amino acid residues and adopts a distinctive ␤-sandwich motif. Despite the lack of amino acid sequence homology, the overall topology of the ␤-sandwich is similar to that first observed in domain 5 of ␤-galactosidase from E. coli (11). This ␤-sheet architecture has since been seen in the central domain of copper amine oxidase (12), the C-te...

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Section: Minireview: Enzymes Of the Leloir Pathway 43886mentioning

confidence: 99%

Structure and Function of Enzymes of the Leloir Pathway for Galactose Metabolism

Holden

Rayment

Thoden

2003

Journal of Biological Chemistry

441

335

View full text Add to dashboard Cite

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“…5). Finally, although R201H, L139P, E291K, and Y323D have all been identified in samples derived from patients with galac- tosemia, no data have been reported assigning specific GALT activity levels to these substitutions (9,44).…”

Section: Allelementioning

confidence: 99%

Relationship between Genotype, Activity, and Galactose Sensitivity in Yeast Expressing Patient Alleles of Human Galactose-1-phosphate Uridylyltransferase

Riehman

Crews

Fridovich‐Keil³

2001

Journal of Biological Chemistry

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Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT) results in the potentially lethal disorder galactosemia; the biochemical basis of pathophysiology in galactosemia remains unknown. We have applied a yeast expression system for human GALT to test the hypothesis that genotype will correlate with GALT activity measured in vitro and with metabolite levels and galactose sensitivity measured in vivo. In particular, we have determined the relative degree of functional impairment associated with each of 16 patient-derived hGALT alleles; activities ranged from null to essentially normal. Next, we utilized strains expressing these alleles to demonstrate a clear inverse relationship between GALT activity and galactose sensitivity. Finally, we monitored accumulation of galactose-1-P, UDP-gal, and UDP-glc in yeast expressing a subset of these alleles. As reported for humans, yeast deficient in GALT, but not their wild type counterparts, demonstrated elevated levels of galactose 1-phosphate and diminished UDP-gal upon exposure to galactose. These results present the first clear evidence in a genetically and biochemically amenable model system of a relationship between GALT genotype, enzyme activity, sensitivity to galactose, and aberrant metabolite accumulation. As such, these data lay a foundation for future studies into the underlying mechanism(s) of galactose sensitivity in yeast and perhaps other eukaryotes, including humans.The enzyme galactose-1-phosphate uridylyltransferase (GALT) 1 catalyzes the second step of the Leloir pathway of galactose metabolism, converting UDP-glucose and galactose 1-phosphate (gal-1-P) to glucose 1-phosphate and UDP-galactose (UDP-gal) (1, 2). Impairment of human GALT (hGALT) results in the potentially lethal disorder classic galactosemia (2, 3).Currently, most infants with classic galactosemia born in industrialized nations are detected in the neonatal period by mandated newborn screening procedures. Dietary restriction of galactose initiated early and maintained throughout life for these patients prevents the potentially lethal sequelae of the disorder. Unfortunately, despite treatment, the long term outcome for these patients is mixed; 85% of girls with galactosemia experience primary ovarian failure, and 30 -50% of patients of both genders demonstrate learning disabilities and speech and/or motor dysfunction, among other complications (4). Although aberrant accumulation or depletion of key galactose metabolites, including gal-1-P, UDP-gal, galactitol, and others are hypothesized as underlying the observed complications (reviewed in Refs. 2 and 3), the biochemical mechanism of pathophysiology in galactosemia remains unknown.

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Galactose Breath Testing Distinguishes Variant and Severe Galactose-1-Phosphate Uridyltransferase Genotypes

et al. 2000

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A galactose breath test that quantitates [1][2][3][4][5][6][7][8][9][10][11][12][13] C]galactose conversion to 13 CO 2 provides information on the whole body galactose oxidative capacity. As there is little information on the relationship between whole body oxidation and the genotype in patients with galactosemia, we measured the 13 CO 2 excretion for 2 h after administration of [1-13 C]galactose in 37 patients (3-48 y old) with galactose-1-phosphate uridyltransferase (GALT) deficiency and 20 control subjects (3-37 y old). Eleven patients with the common Q188R/Q188R genotype and no detectable erythrocyte GALT activity eliminated Ͻ2% of a bolus of [1-13 C]galactose as 13 CO 2 compared with 8.47 to 28.23% in controls. This defines a severe metabolic phenotype. Seven patients with one Q188R allele and a second mutant allele such as L195P, E308K, V151A, M142K, or Q344K and one patient with a K285N/unknown genotype also released Ͻ2% as 13 CO 2 in 2 h. The presence of N314D or S135L as the second mutant allele does not impair total body galactose oxidation, as individuals with the GALT genotype of Q188R/ N314D, K285N/N314D, and Q188R/S135L had normal 2-h galactose breath tests. Subjects with S135L/S135L, N314D/N314D, S135L/⌬T2359 as well as other rarer genotypes such as R258C/ Y209C, E203 K/IVSC-N314D, K285N/T138M, Q188R/D113N, S135L/F171S, R148W/N314D, and IVSC-N314D/N314D oxidized galactose comparable to controls. The dissociation of residual erythrocyte GALT activity and whole body galactose oxidative capacity is exemplified by blacks with a S135L/S135L genotype and absent erythrocyte GALT activity. An oral 2-h [1- Hereditary galactosemia is due to a deficiency in GALT enzyme activity (1). Inherited as an autosomal recessive trait, classic galactosemia is the term frequently used to describe the disease phenotype associated with hepatotoxicity in the infant. The most common genotype producing severe GALT deficiency is the presence of a GALT mutation in which arginine is substituted for glutamine at amino acid 188 (Q188R) (2, 3).However, variant or less severe phenotypes exist even when GALT enzyme activity in erythrocytes is absent. The most notable example is the type in blacks (4, 5) now associated with the S135L allele (6, 7). There is debate as to whether the relatively common Duarte/galactosemia compound heterozygosity state with substantial residual erythrocyte GALT activity (8) due to one N314D allele (9, 10) and one galactosemia allele such as Q188R results in clinical disease. Additionally, the Duarte allele may not decrease enzyme activity by an average of 25% if associated with a 1721 C3 T transition in cis that may increase GALT translation (11,12). As is often the case with nutritional toxicity-genetic diseases, phenotype characterization on clinical and even biochemical grounds is not

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A molecular approach to galactosemia

Cited by 62 publications

References 19 publications

Structure and Function of Enzymes of the Leloir Pathway for Galactose Metabolism

Structure and Function of Enzymes of the Leloir Pathway for Galactose Metabolism

Relationship between Genotype, Activity, and Galactose Sensitivity in Yeast Expressing Patient Alleles of Human Galactose-1-phosphate Uridylyltransferase

Galactose Breath Testing Distinguishes Variant and Severe Galactose-1-Phosphate Uridyltransferase Genotypes

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