Maurice J. Bessman

ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology

Free ADP-ribose (ADPR), a product of NAD hydrolysis and a breakdown product of the calcium-release second messenger cyclic ADPR (cADPR), has no defined role as an intracellular signalling molecule in vertebrate systems. Here we show that a 350-amino-acid protein (designated NUDT9) and a homologous domain (NUDT9 homology domain) near the carboxy terminus of the LTRPC2/TrpC7 putative cation channel both function as specific ADPR pyrophosphatases. Whole-cell and single-channel analysis of HEK-293 cells expressing LTRPC2 show that LTRPC2 functions as a calcium-permeable cation channel that is specifically gated by free ADPR. The expression of native LTRPC2 transcripts is detectable in many tissues including the U937 monocyte cell line, in which ADPR induces large cation currents (designated IADPR) that closely match those mediated by recombinant LTRPC2. These results indicate that intracellular ADPR regulates calcium entry into cells that express LTRPC2.

Studies on the biochemical basis of spontaneous mutation: II. The incorporation of a base and its analogue into DNA by wild-type, mutator and antimutator DNA polymerases

Abstract The incorporation and turnover of adenine and its analogue 2-aminopurine into DNA by purified wild-type, mutator, and antimutator T4 DNA polymerase have been measured. Antimutators incorporate less 2-aminopurine into DNA than does wild type, and imitators incorporate more than wild type. Analysis of these data is consistent with the idea that the incorporation of 2-aminopurine is influenced primarily by the ratio of 3′-exonuclease to polymerase activities of the different enzymes. The experimental results conform to a model equation, which expresses the base incorporation frequency as a function of the polymerase insertion and removal activities. Some of the implications of the model equation are examined in this and the accompanying paper.

Enzymic synthesis of deoxyribonucleic acid

The deoxyribonucleic acid-synthesizing enzyme (polymerase) purified from Escherichiu coli catalyzes the extensive formation of deoxyribonucleic acid when the triphosphates of the four deoxyribonucleosides commonly found in deoxyribonucleic acid are incubated with primer deoxyribonucleic acid (1). In the absence of one, two, or three of the deoxyribonucleoside triphosphates, a very slight reaction occurs which represents the addition of only one or a few deoxyribonucleotide residues to the deoxyribonucleoside end of a deoxyribonucleic acid primer molecule (2). Both reactions occur without lag and both are detectable only in the presence of added deoxyribonucleic acid. This report describes a polymerization reaction in the absence of primer deoxyribonucleic acid catalyzed by the same enzyme preparation. When the four deoxyribonucleoside triphosphates are incubated with the enzyme but without any added deoxyribonucleic acid, a long lag period with no detectable reaction is observed, after which there occurs a rapid synthesis of a polymer which contains only deoxyadenylate and deoxythymidylate. The synthesis of this polymer does not require deoxycytidine triphosphate or deoxyguanosine triphosphate and proceeds in the same manner in their absence. The polymer contains deoxyadenylate and deoxythymidylate in equal proportions and in alternating sequence. As judged by viscometric, sedimentation, and spectrophotometric studies, it is a rigid, double-stranded macromolecule like deoxyribonucleic acid. When this copolymer of deoxyadenylate and deoxythymidylate is used as a primer in the enzymatic reaction with deoxyadenosine triphosphate and deoxythymidine triphosphate, there is a prompt and extensive synthesis of an identical copolymer. Earlier references to these studies have been reported from this laboratory (3, 4).

The structure of ADP-ribose pyrophosphatase reveals the structural basis for the versatility of the Nudix family.

Regulation of cellular levels of ADP-ribose is important in preventing nonenzymatic ADP-ribosylation of proteins. The Escherichia coli ADP-ribose pyrophosphatase, a Nudix enzyme, catalyzes the hydrolysis of ADP-ribose to ribose-5-P and AMP, compounds that can be recycled as part of nucleotide metabolism. The structures of the apo enzyme, the active enzyme and the complex with ADP-ribose were determined to 1.9Å, 2.7Å and 2.3Å, respectively. The structures reveal a symmetric homodimer with two equivalent catalytic sites, each formed by residues of both monomers, requiring dimerization through domain swapping for substrate recognition and catalytic activity. The structures also suggest a role for the residues conserved in each Nudix subfamily. The Nudix motif residues, folded as a loop-helix-loop tailored for pyrophosphate hydrolysis, compose the catalytic center; residues conferring substrate specificity occur in regions of the sequence removed from the Nudix motif. This segregation of catalytic and recognition roles provides versatility to the Nudix family.

NUDT9, a Member of the Nudix Hydrolase Family, Is an Evolutionarily Conserved Mitochondrial ADP-ribose Pyrophosphatase

We have recently characterized the protein product of the human NUDT9 gene as a highly specific ADP-ribose (ADPR) pyrophosphatase (1). We now report an analysis of the human NUDT9 gene and its potential alternative transcripts along with detailed studies of the enzymatic properties and cell biological behavior of human NUDT9 protein. Our analysis of the human NUDT9 gene and twenty-two distinct cloned NUDT9 transcripts indicates that the full-length NUDT9α transcript is the dominant form, and suggests that an alternative NUDT9β transcript (2) occurs as the result of a potentially aberrant splice from a cryptic donor site within the first exon to the splice acceptor site of exon 2. Computer analysis of the predicted protein of the NUDT9α transcript identified an N-terminal signal peptide or subcellular targeting sequence. Using green fluorescence protein tagging, we demonstrate that the predicted human NUDT9α protein is targeted highly specifically to mitochondria, whereas the predicted protein of the NUDT9β transcript, which is missing this sequence, exhibits no clear subcellular localization. Investigation of the physical and enzymatic properties of NUDT9 indicates that it is functional as a monomer, optimally active at near neutral pH, and that it requires divalent metal ions and an intact Nudix motif for enzymatic activity. Furthermore, partial proteolysis of NUDT9 suggests that NUDT9 enzymes consist of two distinct domains: a proteolytically resistant C-terminal domain retaining essentially full specific ADPR pyrophosphatase activity and a proteolytically labile N-terminal portion that functions to enhance the affinity of the C-terminal domain for ADPR.

A NOVEL GDP-MANNOSE MANNOSYL HYDROLASE SHARES HOMOLOGY WITH THE MUTT FAMILY OF ENZYMES

The product of the Escherichia coli orf1.9, or yefc, gene (GenBank accession number L11721) has been expressed under the control of a T7 promoter, purified to apparent homogeneity, and identified as a novel enzyme that hydrolyzes GDP-mannose or GDP-glucose to GDP and the respective hexose. The enzyme has little or no activity on other nucleotides, dinucleotides, nucleotide sugars, or sugar phosphates. It has a pH optimum between 9.0 and 9.5, a K of 0.3 mM, and a Vmax of 1.6 μmol min mg for GDP-mannose, and it requires divalent cations for activity. This enzyme of 160 amino acids (M = 18, 405) contains the consensus sequence GX(I/L/V)(E/Q)(X)ET(X)R(X)E(X)(I/L), characteristic of the MutT family of proteins and previously shown to form part of the nucleotide-binding site of MutT (Frick, D. N., Weber, D. J., Abeygunawardana, C., Gittis, A. G., Bessman, M. J., and Mildvan, A. S.(1995) Biochemistry 34, 5577-5586). A comparison of the enzymatic reactions catalyzed by the GDP-mannose mannosyl hydrolase and the other enzymes of the MutT family suggests that the consensus signature sequence designates a novel nucleoside diphosphate binding site and catalytic motif.

The Gene, ialA, Associated with the Invasion of Human Erythrocytes by Bartonella bacilliformis, Designates a Nudix Hydrolase Active on Dinucleoside 5′-Polyphosphates

ialA, one of two genes associated with the invasion of human red blood cells by Bartonella bacilliformis, the causative agent of several diseases, has been cloned and expressed in Escherichia coli. The protein, IalA, contains an amino acid array characteristic of a family of enzymes, the Nudix hydrolases, active on a variety of nucleoside diphosphate derivatives. IalA has been purified, identified, and characterized as an enzyme catalyzing the hydrolysis of members of a class of signaling nucleotides, the dinucleoside polyphosphates, with its highest activity on adenosine 5′-tetraphospho-5′-adenosine (Ap4A), but also hydrolyzing Ap5A, Ap6A, Gp4G, and Gp5G. In each case, a pyrophosphate linkage is cleaved yielding a nucleoside triphosphate and the remaining nucleotide moiety.

Studies on the biochemical basis of spontaneous mutation: V. Effect of temperature on mutation frequency☆☆☆

Abstract Temperature, in the range of 15 °C to 40 °C, has a pronounced effect on the incorporation of 2-aminopurine deoxynucleotides into DNA by purified bacteriophage T4-induced DNA polymerase. Whereas the total rate of utilization of the 2-aminopurine deoxynucleoside triphosphate increases with increasing temperature, a greater proportion is converted to the monophosphate by the editing nuclease of the enzyme. Therefore, the amount of analogue incorporated goes through a maximum and then decreases with increasing temperature. These results, obtained in vitro , have been correlated with effects of temperature on 2-aminopurine induced and spontaneous mutation rates of several r II markers, and they have been generalized to an hypothesis which holds that the stability of the helix immediately preceding the incoming nucleotide is an important factor in determining the accuracy of DNA replication. We suggest that there is a higher probability of making errors via base substitutions in a more stable (G + C-rich) rather than a less stable (A + T-rich) microenvironment.

Characterization of the mutX gene of Streptococcus pneumoniae as a homologue of Escherichia coli mutT, and tentative definition of a catalytic domain of the dGTP pyrophosphohydroiases

We show that deletion of a gene of Streptococcus pneumoniae, which we call mutX, confers a mutator phenotype to resistance to streptomycin. Analysis of the DNA sequence changes that occurred in several streptomycin‐resistant mutants showed that mutations are unidirectional AT to CG transversions. The mutX gene is located immediately downstream of the previously identified ung gene and genetic evidence suggests that the two genes are coordinately regulated. Nucleotide sequence determination reveals that the mutX gene encodes a 17870 Da protein (154 residues) which exhibits significant homology with the MutT protein of Escherichia coli, a nucleoside tri‐phosphatase (dGTP pyrophosphohydrolase). The mutX gene complements the E coli mutT mutator phenotype when introduced on a plasmid. Site‐directed mutagenesis and analysis of nitrosoguanidine‐induced mutT mutants suggest that a small region of high homology between the two proteins (61% identity over 23 residues) is part of the catalytic site of the nucleoside triphosphatase. Computer searching for sequence homology to MutX uncovered a second E. coli protein, the product of orf17, a gene of unknown function located near the ruvC gene. The region of high homology between MutX and MutT is also conserved in this protein, which raises the interesting possibility that the orf17 gene plays some role in determining mutation rates in E. coli. Finally, a small set of proteins, including a family of virus‐encoded proteins and two evolutionarily conserved proteins encoded by an antisense transcript from the Xenopus laevis and human bFGF genes, were also found to harbour significant homology to this highly conserved region.

The Nudix hydrolases of Deinococcus radiodurans

All 21 of the Nudix hydrolase genes from the radiation‐resistant organism Deinococcus radiodurans have been cloned into vectors under the control of T7 promoters and expressed as soluble proteins in Escherichia coli. Their sizes range from 9.8 kDa (91 amino acids) to 59 kDa (548 amino acids). Two novel proteins were identified, each with two Nudix boxes in its primary structure, unique among all other known Nudix hydrolases. Extracts of each of the expressed proteins were assayed by a generalized procedure that measures the hydrolysis of nucleoside diphosphate derivatives, and several enzymatic activities were tentatively identified. In addition to representatives of known Nudix hydrolase subfamilies active on ADP‐ribose, NADH, dinucleoside polyphosphates or (deoxy)nucleoside triphosphates, two new enzymes, a UDP‐glucose pyrophosphatase and a CoA pyrophosphatase, were identified.