Galactose-1-phosphate uridylytransferase. Purification of the enzyme and stereochemical course of each step of the double-displacement mechanism

Arabshahi, Abolfazl; Rs, Brody; Smallwood, Angela; Tsai, TC; Pa, Frey

doi:10.1021/bi00367a036

Cited by 30 publications

(28 citation statements)

References 18 publications

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“…In the cascade of activation steps for glutamine synthetase, for example, the activity of P II protein is controlled by its degree of uridylylation, which in turn controls glutamine synthetase activity (1,28). Furthermore, the interconversion of galactose-1-phosphate and glucose-1-phosphate occurs in their uridylylated forms, using uridylyl transferase as a covalent intermediate (5,16). In the P II protein, a tyrosine residue serves as the acceptor for the uridylate residue (1,59), whereas with uridyl transferase in galactose metabolism, a histidine is the uridylate acceptor (64).…”

Section: Discussionmentioning

confidence: 99%

Intramolecular and Intermolecular Uridylylation by Poliovirus RNA-Dependent RNA Polymerase

Richards

Spagnolo

Lyle

et al. 2006

J Virol

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The 22-amino-acid protein VPg can be uridylylated in solution by purified poliovirus 3D polymerase in a template-dependent reaction thought to mimic primer formation during RNA amplification in infected cells. In the cell, the template used for the reaction is a hairpin RNA termed 2C-cre and, possibly, the poly(A) at the 3 end of the viral genome. Here, we identify several additional substrates for uridylylation by poliovirus 3D polymerase. In the presence of a 15-nucleotide (nt) RNA template, the poliovirus polymerase uridylylates other polymerase molecules in an intermolecular reaction that occurs in a single step, as judged by the chirality of the resulting phosphodiester linkage. Phosphate chirality experiments also showed that VPg uridylylation can occur by a single step; therefore, there is no obligatory uridylylated intermediate in the formation of uridylylated VPg. Other poliovirus proteins that could be uridylylated by 3D polymerase in solution were viral 3CD and 3AB proteins. Strong effects of both RNA and protein ligands on the efficiency and the specificity of the uridylylation reaction were observed: uridylylation of 3D polymerase and 3CD protein was stimulated by the addition of viral protein 3AB, and, when the template was poly(A) instead of the 15-nt RNA, the uridylylation of 3D polymerase itself became intramolecular instead of intermolecular. Finally, an antiuridine antibody identified uridylylated viral 3D polymerase and 3CD protein, as well as a 65-to 70-kDa host protein, in lysates of virus-infected human cells.Many positive-sense single-stranded RNA viral genomes are relatively small templates that encode information for complex viral replication cycles and thus require highly efficient utilization of limited coding capacity. The number of functional activities expressed from any genome can be expanded by utilizing both precursor polypeptides and their processed cleavage products. For example, poliovirus protein 3D is an RNA-dependent RNA polymerase, while its presumed precursor, 3CD, which is a fusion between the 3C protease and 3D polymerase, manifests no polymerase activity but functions as a specific protease with substrate recognition properties different from its cleavage product, 3C protease (29, 68). Another mechanism that expands coding capacity is the utilization of the same polypeptide for multiple functions. For example, in addition to proteolytic activity, 3CD also functions as a specific RNAbinding protein with crucial roles in viral RNA replication. It binds the 5Ј-terminal RNA cloverleaf structure (2, 3, 23, 49) as well as an internal stem-loop structure in the 2C coding region (66, 67); both interactions are required for the initiation of RNA replication. Finally, posttranslational modifications can further modify the function of viral proteins; for example, the covalent myristoylation of viral capsid protein VP0 facilitates its transition from a precursor protein to a component of an assembled capsid (4, 42).The poliovirus genome contains a single open reading frame that c...

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Section: Discussionmentioning

confidence: 99%

Intramolecular and Intermolecular Uridylylation by Poliovirus RNA-Dependent RNA Polymerase

Richards

Spagnolo

Lyle

et al. 2006

J Virol

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“…The most common form of galactosemia arises from defects in galactose-1-phosphate uridylyltransferase and as such it has been the subject of intensive kinetic and structural investigations for many years (Holden et al 2003). Studies have demonstrated that the reaction mechanism of this enzyme proceeds through a covalently bound intermediate via a double displacement pathway (Arabshahi et al 1986). …”

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confidence: 99%

The molecular architecture of glucose‐1‐phosphate uridylyltransferase

Thoden

Holden

2007

Protein Science

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Glucose-1-phosphate uridylyltransferase, also referred to as UDP-glucose pyrophosphorylase or UGPase, catalyzes the formation of UDP-glucose from glucose-1-phosphate and UTP. Not surprisingly, given the central role of UDP-glucose in glycogen synthesis and in the production of glycolipids, glycoproteins, and proteoglycans, the enzyme is ubiquitous in nature. Interestingly, however, the prokaryotic and eukaryotic forms of the enzyme are unrelated in amino acid sequence and structure. Here we describe the cloning and structural analysis to 1.9 Å resolution of the UGPase from Escherichia coli. The protein is a tetramer with 222 point group symmetry. Each subunit of the tetramer is dominated by an eight-stranded mixed b-sheet. There are two additional layers of b-sheet (two and three strands) and 10 a-helices. The overall fold of the molecule is remarkably similar to that observed for glucose-1-phosphate thymidylyltransferase in complex with its product, dTDP-glucose. On the basis of this similarity, a UDP-glucose moiety has been positioned into the active site of UGPase. This protein/product model predicts that the side chains of Gln 109 and Asp 137, respectively, serve to anchor the uracil ring and the ribose of UDP-glucose to the protein. The b-phosphoryl group of the product is predicted to lie within hydrogen bonding distance to the e-nitrogen of Lys 202 whereas the carboxylate group of Glu 201 is predicted to bridge the 29-and 39-hydroxyl groups of the glucosyl moiety. Details concerning the overall structure of UGPase and a comparison with glucose-1-phosphate thymidylyltransferase are presented.Keywords: UDP-glucose; glucose-1-phosphate uridylyltransferase; UDP-glucose pyrophosphorylase; X-ray structure; carbohydrates The conversion of b-D-galactose to the more metabolically useful glucose-1-phosphate is accomplished in most organisms by the action of four enzymes that constitute the Leloir pathway (Holden et al. 2003). In the first step of this pathway, b-D-galactose is epimerized to a-D-galactose by galactose mutarotase. The next step involves the ATP-dependent phosphorylation of a-Dgalactose by galactokinase to yield galactose-1-phosphate. Subsequently, in the third step, the enzyme 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 UDPgalactose 4-epimerase. In humans, mutations in the genes that encode the galactokinase, the galactose-1-phosphate uridylyltransferase, or the epimerase can result in the diseased state referred to as galactosemia with clinical manifestations including intellectual retardation, speech disorders, liver dysfunction, and cataract formation. The most common form of galactosemia arises from defects in galactose-1-phosphate uridylyltransferase and as such it has been the subject of intensive kinetic and structural investigations for many years (Holden et al. 2003). Studies have...

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“…In the second-half reaction, Gal-1-P enters the active site cleft and attacks the uridylylated nitrogen on the imidazole ring of the active site histidine, forming UDP-Gal. This second product then dissociates, regenerating free enzyme (2,7).…”

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confidence: 99%

Covalent Heterogeneity of the Human Enzyme Galactose-1-phosphate Uridylyltransferase

Henderson

Wells²,

Fridovich‐Keil

2000

Journal of Biological Chemistry

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Galactose-1-phosphate uridylyltransferase (GALT) acts by a double displacement mechanism, catalyzing the second step in the Leloir pathway of galactose metabolism. Impairment of this enzyme results in the potentially lethal disorder, galactosemia. Although the microheterogeneity of native human GALT has long been recognized, the biochemical basis for this heterogeneity has remained obscure. We have explored the possibility of covalent GALT heterogeneity using denaturing twodimensional gel electrophoresis and Western blot analysis to fractionate and visualize hemolysate hGALT, as well as the human enzyme expressed in yeast. In both contexts, two predominant GALT species were observed. To define the contribution of uridylylated enzyme intermediate to the two-spot pattern, we exploited the null allele, H186G-hGALT. The Escherichia coli counterpart of this mutant protein (H166G-eGALT) has previously been demonstrated to fold properly, although it cannot form covalent intermediate. Analysis of the H186G-hGALT protein demonstrated a single predominant species, implicating covalent intermediate as the basis for the second spot in the wild-type pattern. In contrast, three naturally occurring mutations, N314D, Q188R, and S135L-hGALT, all demonstrated the twospot pattern. Together, these data suggest that uridylylated hGALT comprises a significant fraction of the total GALT enzyme pool in normal human cells and that three of the most common patient mutations do not disrupt this distribution.Galactose-1-phosphate uridylyltransferase (GALT) 1 catalyzes the second step of the Leloir pathway of galactose metabolism, converting UDP-glucose (UDP-Glc) and galactose-1-phosphate (Gal-1-P) into glucose-1-phosphate (Glc-1-P) and UDP-galactose (UDP-Gal) (Fig. 1). Impairment of human GALT results in the potentially lethal disorder galactosemia. Both the bacterial and human GALT enzymes function as homodimers (1, 2) and display ping-pong kinetics with catalysis that proceeds via a covalent intermediate (3,4). In the first-half reaction, a histidine residue at the active site (His-166 in Escherichia coli GALT and His-186 in the human enzyme) serves as a nucleophile, attacking the ␣-phosphorus of UDP-Glc. Glc-1-P is released, leaving behind a uridylylated enzyme intermediate (5,6). In the second-half reaction, Gal-1-P enters the active site cleft and attacks the uridylylated nitrogen on the imidazole ring of the active site histidine, forming UDP-Gal. This second product then dissociates, regenerating free enzyme (2, 7).It has been known for more than 30 years that human GALT derived from red blood cells or other tissues is heterogeneous when fractionated under native conditions and visualized with an activity overlay stain (8). Studies using starch, agarose, or acrylamide isoelectric focusing gels have revealed a complex pattern of up to six bands (9 -12). Additionally, specific variants of human GALT, such as the Duarte and Los Angeles alleles, have been characterized in part by their shifted banding patterns (8). Despite its common ob...

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Galactose-1-phosphate uridylytransferase. Purification of the enzyme and stereochemical course of each step of the double-displacement mechanism

Cited by 30 publications

References 18 publications

Intramolecular and Intermolecular Uridylylation by Poliovirus RNA-Dependent RNA Polymerase

Intramolecular and Intermolecular Uridylylation by Poliovirus RNA-Dependent RNA Polymerase

The molecular architecture of glucose‐1‐phosphate uridylyltransferase

Covalent Heterogeneity of the Human Enzyme Galactose-1-phosphate Uridylyltransferase

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