High Resolution X-ray Structure of Galactose Mutarotase from Lactococcus lactis

Thoden, James B.; Holden, Hazel M.

doi:10.1074/jbc.m201415200

Cited by 42 publications

(60 citation statements)

References 23 publications

(30 reference statements)

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“…The X-ray structure of galactose mutarotase from L. lactis at high resolution reveals that the residues Arg 71 , His 96 , His 170 , Asp 243 and Glu 304 are responsible for anchoring the sugar to GalM. In 34 GalM sequences in SWISS-PROT, a histidine residue is generally located at the centre region and another histidine residue is~60-80 aa distant from the centre one at the N terminus (Thoden & Holden, 2002). For instance, in E. coli, the mutation of the centre histidine, His 175 , results in complete loss of enzyme activity, and in L. lactis, mutation of either the His 96 or the His 170 residue can lead to a significant decrease in enzyme activity (Beebe & Frey, 1998).…”

Section: Discussionmentioning

confidence: 99%

“…Mutarotase (GalM; EC 5.1.3.3) is a functional enzyme in many bacteria (Brahma & Bhattacharyya, 2004;Majumdar et al, 2004;Poolman et al, 1990;Thoden & Holden, 2002), and catalyses the interconversion of the a-and b-anomers of galactose and glucose. Also, mutarotase is one of the key elements in many gal operons (Bettenbrock & Alpert, 1998;Thoden & Holden, 2002;Vaillancourt et al, 2002;Vaughan et al, 2001). Nevertheless, on the basis of the current genomic data for T. tengcongensis, there is no mutarotase gene.…”

Section: Functional Annotation Of the Gal Proteins In T Tengcongensismentioning

confidence: 99%

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Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

et al. 2009

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On the basis of the Thermoanaerobacter tengcongensis genome, a novel type of gal operon was deduced. The gene expression and biochemical properties of this operon were further characterized. RT-PCR analysis of the intergenic regions suggested that the transcription of the gal operon was continuous. With gene cloning and enzyme activity assays, TTE1929, TTE1928 and TTE1927 were identified to be GalT, GalK and GalE, respectively. Results elicited from polarimetry assays revealed that TTE1925, a hypothetical protein, was a novel mutarotase, termed MR-Tt. TTE1926 was identified as a regulator that could bind to two operators in the operon promoter. The transcriptional start sites were mapped, and this suggested that there are two promoters in this operon. Expression of the gal genes was significantly induced by galactose, whereas only MR-Tt expression was detected in glucose-cultured T. tengcongensis at both the mRNA and the protein level. In addition, the abundance of gal proteins was examined at different temperatures. At temperatures ranging from 60 to 80 6C, the level of MR-Tt protein was relatively stable, but that of the other gal proteins was dramatically decreased. The operator-binding complexes were isolated and identified by electrophoretic mobility shift assay-liquid chromatography (EMSA-LC) MS-MS, which suggested that several regulatory proteins, such as GalR and a sensory histidine kinase, participate in the regulation of the gal operon.

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Section: Discussionmentioning

confidence: 99%

Section: Functional Annotation Of the Gal Proteins In T Tengcongensismentioning

confidence: 99%

Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

et al. 2009

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“…The first three-dimensional structure of a dimeric mutarotase, namely that from Lactococcus lactis, was reported in 2002 (24). The enzyme was shown to adopt a ␤-sandwich motif, similar to that seen in domain 5 of ␤-galactosidase (25) with the sugar binding site located in a wide, shallow cleft.…”

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confidence: 99%

“…The preference of these enzymes for galactose over glucose was addressed structurally with the L. lactis mutarotase and, at least in the case of this bacterial protein, is explained by the existence of nonproductive binding modes in the active site for glucose and its derivatives (33). With respect to quaternary structure, human mutarotase appears to be monomeric (10,30), whereas the L. lactis enzyme was shown to be a dimer on the basis of ultracentrifugation experiments (24). The biochemical significance, if any, of these differences in quaternary structure is not understood at the present time.…”

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confidence: 99%

“…Five highly conserved residues (Arg-71, His-96, His-170, Asp-243, and Glu-304) were within hydrogen-bonding distance to the hydroxyl groups of the bound galactose ligand. Previous kinetic studies suggested that the reaction mechanism for the enzyme proceeds via an acid/base mechanism with a transient ring opening (26,27), and the model of the mutarotase from L. lactis implicated Glu-304 as the likely catalytic base and either His-96 or His-170 as the catalytic acid (24). Accordingly, the reaction mechanism of galactose mutarotase is thought to occur via abstraction of the proton from the C-1 hydroxyl group by Glu-304 in the L. lactis enzyme and protonation of the ring oxygen of the sugar by His-170, which results in ring opening.…”

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Molecular Structure of Human Galactose Mutarotase

Thoden

Timson

Reece

et al. 2004

Journal of Biological Chemistry

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Galactose mutarotase catalyzes the conversion of ␤-Dgalactose to ␣-D-galactose during normal galactose metabolism. The enzyme has been isolated from bacteria, plants, and animals and is present in the cytoplasm of most cells. Here we report the x-ray crystallographic analysis of human galactose mutarotase both in the apoform and complexed with its substrate, ␤-D-galactose. The polypeptide chain folds into an intricate array of 29 ␤-strands, 25 classical reverse turns, and 2 small ␣-helices. There are two cis-peptide bonds at Arg-78 and Pro-103. The sugar ligand sits in a shallow cleft and is surrounded by Asn-81, Arg-82, His-107, His-176, Asp-243, Gln-279, and Glu-307. Both the side chains of Glu-307 and His-176 are in the proper location to act as a catalytic base and a catalytic acid, respectively. These residues are absolutely conserved among galactose mutarotases. To date, x-ray models for three mutarotases have now been reported, namely that described here and those from Lactococcus lactis and Caenorhabditis elegans. The molecular architectures of these enzymes differ primarily in the loop regions connecting the first two ␤-strands. In the human protein, there are six extra residues in the loop compared with the bacterial protein for an approximate longer length of 9 Å. In the C. elegans protein, the first 17 residues are missing, thereby reducing the total number of ␤-strands by one.During normal galactose metabolism, ␤-D-galactose is converted to glucose 1-phosphate via the action of four enzymes that constitute the Leloir pathway as shown in Scheme 1 (1). In the first step of this pathway, ␤-D-galactose is epimerized to ␣-D-galactose through the action of galactose mutarotase. The second step involves the phosphorylation of ␣-D-galactose to galactose 1-phosphate by galactokinase. As indicated in Scheme 1, galactose 1-phosphate uridylyltransferase catalyzes the third step by transferring a UMP group from UDP-glucose to galactose 1-phosphate, thereby generating glucose 1-phosphate and UDP-galactose. To complete the pathway, UDPgalactose is converted to UDP-glucose by UDP-galactose 4-epimerase.Mutations in three of the enzymes of the Leloir pathway, namely galactokinase, galactose 1-phosphate uridylyltransferase, or UDP-galactose 4-epimerase, have been demonstrated to result in the diseased state known as galactosemia (2, 3). The symptoms of this genetic disease include early onset cataracts (typically within the first two years of life) and, in more severe cases, liver, kidney, and brain damage. Cataract formation is believed to be caused by the buildup of unmetabolized galactose in the lens of the eye. Aldose reductase catalyzes the reduction of the sugar to galactitol (dulcitol), which is not, in contrast to galactose, readily transported across the plasma membrane (4 -6). As such, the accumulation of this highly osmotically active compound draws excess water into the lens resulting in cataracts by a mechanism that is not fully understood. The more severe symptoms of galactosemia are most likely caused b...

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Through the Looking Glass: Chiral Recognition of Substrates and Products at the Active Sites of Racemases and Epimerases

Bearne

2020

Chemistry A European J

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Unlike most enzymes, which exhibit stereospecific substrate binding, racemases and epimerases bind and catalyze the reversible interconversion of enantiomeric and epimeric pairs of substrates. Over the past 15 years, a growing number of racemase and epimerase structures have been solved, furnishing insights into the nature of chiral recognition of substrates by these enzymes. Those enzymes catalyzing stereoinversion of a carbon acid substrate through a direct 1,1‐proton transfer mechanism all bind their substrates in a mirror‐image packing orientation. This does not apply generally to racemases and epimerases that use other mechanisms, such as NADH‐dependent epimerases that employ a “flipping” mechanism. In general, polar groups are bound and fixed at the three binding determinants on the protein defining a pseudo‐mirror plane, while nonpolar groups may be mobile. The hydrogen atoms on each stereocenter are positioned antipodal with respect to the pseudo‐mirror plane, making a two‐base mechanism imperative. Recognition that mirror‐image packing is the common binding mode for enantiomeric or epimeric substrates of these enzymes should inform modelling/docking studies and protein engineering.

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High Resolution X-ray Structure of Galactose Mutarotase from Lactococcus lactis

Cited by 42 publications

References 23 publications

Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

Molecular Structure of Human Galactose Mutarotase

Through the Looking Glass: Chiral Recognition of Substrates and Products at the Active Sites of Racemases and Epimerases

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