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Structure and mechanism of an ADP-glucose phosphorylase from Arabidopsis thaliana.

ABSTRACT: The X-ray crystal structure of the At5g18200.1 protein has been determined to a nominal resolution of 2.30 A. The structure has a histidine triad (HIT)-like fold containing two distinct HIT-like motifs. The sequence of At5g18200.1 indicates a distant family relationship to the Escherichia coli galactose-1-P uridylyltransferase (GalT): the determined structure of the At5g18200.1 protein confirms this relationship. The At5g18200.1 protein does not demonstrate GalT activity but instead catalyzes adenylyl transfer in the reaction of ADP-glucose with various phosphates. The best acceptor among those evaluated is phosphate itself; thus, the At5g18200.1 enzyme appears to be an ADP-glucose phosphorylase. The enzyme catalyzes the exchange of (14)C between ADP-[(14)C]glucose and glucose-1-P in the absence of phosphate. The steady state kinetics of exchange follows the ping-pong bi-bi kinetic mechanism, with a k(cat) of 4.1 s(-)(1) and K(m) values of 1.4 and 83 microM for ADP-[(14)C]glucose and glucose-1-P, respectively, at pH 8.5 and 25 degrees C. The overall reaction of ADP-glucose with phosphate to produce ADP and glucose-1-P follows ping-pong bi-bi steady state kinetics, with a k(cat) of 2.7 s(-)(1) and K(m) values of 6.9 and 90 microM for ADP-glucose and phosphate, respectively, at pH 8.5 and 25 degrees C. The kinetics are consistent with a double-displacement mechanism that involves a covalent adenylyl-enzyme intermediate. The X-ray crystal structure of this intermediate was determined to 1.83 A resolution and shows the AMP group bonded to His(186). The value of K(eq) in the direction of ADP and glucose-1-P formation is 5.0 at pH 7.0 and 25 degrees C in the absence of a divalent metal ion, and it is 40 in the presence of 1 mM MgCl(2).

SUBMITTER: McCoy JG

PROVIDER: S-EPMC2532824 | biostudies-literature | 2006 Mar

REPOSITORIES: biostudies-literature

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Structure and mechanism of an ADP-glucose phosphorylase from Arabidopsis thaliana.

McCoy Jason G JG Arabshahi Abolfazl A Bitto Eduard E Bingman Craig A CA Ruzicka Frank J FJ Frey Perry A PA Phillips George N GN

Biochemistry 20060301 10

The X-ray crystal structure of the At5g18200.1 protein has been determined to a nominal resolution of 2.30 A. The structure has a histidine triad (HIT)-like fold containing two distinct HIT-like motifs. The sequence of At5g18200.1 indicates a distant family relationship to the Escherichia coli galactose-1-P uridylyltransferase (GalT): the determined structure of the At5g18200.1 protein confirms this relationship. The At5g18200.1 protein does not demonstrate GalT activity but instead catalyzes ad ...[more]

PMID: 16519510

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Project description:Proteins from the poly(ADP-ribose) polymerase (PARP) family, such as PARP1 and PARP2, use NAD+ as a substrate to catalyze the synthesis of polymeric chains consisting of ADP-ribose units covalently attached to an acceptor molecule. PARP1 and PARP2 are viewed as DNA damage sensors that, upon binding to strand breaks, poly(ADP-ribosyl)ate themselves and nuclear acceptor proteins. The flowering plant Arabidopsis thaliana contains three genes encoding homologs of mammalian PARPs: atPARP1, atPARP2, and atPARP3. Both atPARP1 and atPARP2 contain poly(ADP-ribosyl)ating activity; however, it is unknown whether they could covalently modify DNA by ADP-ribosylating the strand break termini. Here, we report that similar to their mammalian counterparts, the plant atPARP1 and atPARP2 proteins ADP-ribosylate 5'-terminal phosphate residues in duplex DNA oligonucleotides and plasmid containing at least two closely spaced DNA strand breaks. AtPARP1 preferentially catalyzes covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 5'-phosphate, whereas atPARP2 preferentially ADP-ribosylates the nicked and gapped DNA duplexes containing the terminal 5'-phosphate. Similar to their mammalian counterparts, the plant PARP-catalyzed DNA ADP-ribosylation is particularly sensitive to the distance that separates two strand breaks in the same DNA molecule, 1.5 and 1 or 2 turns of helix for atPARP1 and atPARP2, respectively. PAR glycohydrolase (PARG) restored native DNA structure by hydrolyzing the PAR-DNA adducts generated by atPARPs. Biochemical and mass spectrometry analyses of the PAR-DNA adducts showed that atPARPs utilize phosphorylated DNA termini as an alternative to protein acceptor residues to catalyze PAR chain synthesis via phosphodiester bond formation between C1' of ADP-ribose and a phosphate residue of the terminal nucleotide in DNA fragment. Taken together, these data establish the presence of a new type of DNA-modifying activity in Arabidopsis PARPs, suggesting a possible role of DNA ADP-ribosylation in DNA damage signaling and repair of terrestrial plants.

Dataset Information

Structure and mechanism of an ADP-glucose phosphorylase from Arabidopsis thaliana.

Publications

Structure and mechanism of an ADP-glucose phosphorylase from Arabidopsis thaliana.

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