PubMed Health⌕ Search

Biomedical subjects

M J Bessman

Publications and source records attributed to M J Bessman.

At least 19 recordsLinked to original sources
PubMed1995

Solution structure of the MutT enzyme, a nucleoside triphosphate pyrophosphohydrolase.

The MutT enzyme (129 residues) catalyzes the hydrolysis of normal and mutagenic nucleoside triphosphates, such as 8-oxo-dGTP, by substitution at the rarely attacked beta-P, to yield NMP and pyrophosphate. Previous heteronuclear NMR studies of MutT have shown the secondary structure to consist of a five-stranded mixed beta-sheet connected by the loop I-alpha-helix I--loop II motif, by two tight turns, and by loop III, and terminated by loop IV--alpha-helix II [Abeygunawardana et al. (1993) Biochemistry 32, 13071-13080; Weber et al. (1993) Biochemistry 32, 13081-13087). Complete side-chain assignments of 1H and 13C resonances have now been made by 3D C(CO)NH and HCCH-TOCSY experiments. A total of 1461 interproton proximities (11 per residue), obtained by 3D 15N-resolved NOESY-HSQC and 3D 13C-resolved NOESY-HSQC spectra, including 372 long-range NOEs, as well as 65 dihedral angle (phi) restraints and 34 backbone hydrogen bond restraints were used to determine the tertiary structure of MutT by distance geometry, simulated annealing, and energy minimization with the program X-PLOR. The structure is globular and compact with the parallel portion of the beta-sheet sandwiched between the two alpha-helices, forming an alpha+beta fold. The essential divalent cation has previously been shown to bind near residues Gly-37, Gly-38, Lys-39, and Glu-57, and nucleotides have been shown to bind near residues Leu-54 and Val-58 by NMR relaxation methods [Frick et al. (1995) Biochemistry 34, 5577-5586].(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1995

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 Km of 0.3 mM, and a Vmax of 1.6 mumol min-1 mg-1 for GDP-mannose, and it requires divalent cations for activity. This enzyme of 160 amino acids (M(r) = 18, 405) contains the consensus sequence GX(I/L/V)(E/Q)(X)2ET(X)6R(X)4E(X)2(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.

Amino Acid Sequence↗
PubMed1995

NMR studies of the conformations and location of nucleotides bound to the Escherichia coli MutT enzyme.

The MutT enzyme catalyzes the hydrolysis of nucleoside triphosphates to nucleoside monophosphates and pyrophosphate by substitution at the rarely attacked beta-phosphorus. Nucleotides containing bulky substituents at the 8 position of the purine ring are preferentially hydrolyzed. The conformation of the MutT-bound nonhydrolyzable substrate analog Mg(2+)-AMPCPP, determined by 10 intramolecular NOEs and molecular dynamics refinement using a full relaxation matrix analysis with back-calculation of the NOESY intensities, is high anti (chi = 53 +/- 9 degrees), with a C2'-exo, O1'-endo sugar pucker. Similarly, the product of dGTP hydrolysis, dGMP, also binds MutT in a high anti (chi = 73 +/- 9 degrees) C1'-endo conformation based on seven intramolecular NOEs. Such high anti rotations of the base would allow MutT to accommodate nucleotides substituted at the C-8 position with no intramolecular clashes. Changes in chemical shifts in the 1H-15N spectra of the enzyme induced by Mg2+ and Mg2+ AMPCPP suggest that the metal activator and nucleotide interact with residues in loop I, at the carboxyl end of helix I, loop II, loop III, and beta-strands A and B of the secondary structure of MutT. The displacement of Mg2+ by Mn2+ causes the selective disappearance due to paramagnetic broadening of 1H-15N cross peaks from G37, G38, and K39 in loop I and E57 in helix I. Eleven intermolecular NOEs between Mg2+AMPCPP and hydrophobic residues of MutT are found, three of which are tentatively assigned to L67 in loop II and three to L54 in helix I. Similarly, seven intermolecular NOEs between dGMP and hydrophobic residues of the enzyme are found, four of which are tentatively assigned to L54 and two to V58, both in helix I. These interactions indicate that the loop I-helix I-loop II motif contributes significantly to the active site of MutT in accord with mutagenesis studies and with sequence homologies among MutT-like NTP pyrophosphohydrolases.

Adenosine Triphosphate↗
PubMed1995

Cloning, purification, and properties of a novel NADH pyrophosphatase. Evidence for a nucleotide pyrophosphatase catalytic domain in MutT-like enzymes.

An Escherichia coli open reading frame containing significant homology to the active site of the MutT enzyme codes for a novel dinucleotide pyrophosphatase. The motif shared by these two proteins and several others is conserved throughout nature and may designate a nucleotide-binding or pyrophosphatase domain. The E. coli NADH pyrophosphatase has been cloned, overexpressed, and purified to near homogeneity. The protein contains 257 amino acids (M(r) = 29,774) and migrates on gel filtration columns as an apparent dimer. The enzyme catalyzes the hydrolysis of a broad range of dinucleotide pyrophosphates, but uniquely prefers the reduced form of NADH. The Vmax/Km for NADH (69 mumol min-1 mg-1 mM-1) is an order of magnitude higher than for any other dinucleotide pyrophosphate tested. In addition, the Km for NADH (0.1 mM) is 50-fold lower than the Km for NAD+. The hydrolysis of dinucleotide pyrophosphates requires divalent metal ions and yields two mononucleoside 5'-phosphates. The metals that most efficiently stimulate activity are Mg2+ and Mn2+. Although these metals support similar Vmax values at optimal metal concentration, the apparent Km for Mg2+ is 3.7 mM (at 1 mM NADH), whereas the apparent Km for Mn2+ at the same NADH concentration is 30 microM.

Amino Acid Sequence↗
PubMed1994

Purification of the MutX protein of Streptococcus pneumoniae, a homologue of Escherichia coli MutT. Identification of a novel catalytic domain for nucleoside triphosphate pyrophosphohydrolase activity.

The mutX gene of Streptococcus pneumoniae, a homologue of the Escherichia coli mutT mutator gene (Méjean, V., Salles, C., Bullions, L. C., Bessman, M. J., and Claverys, J.-P. (1993) Mol. Microbiol. 11, 323-330) has been cloned into an expression vector, and its gene product, the MutX protein, has been purified to apparent homogeneity. Like MutT, the pure MutX protein hydrolyzes all of the canonical nucleoside triphosphates at different rates with a preference for dGTP, yielding nucleoside monophosphates and inorganic pyrophosphate. Despite this similarity in enzymatic activity, the two proteins have notably dissimilar primary and quaternary structures. They share only a small region of amino acid homology, and under the same conditions in which MutT exists as a monomer in solution, MutX behaves as a trimer. The small region of conserved amino acid sequence most likely identifies a protein domain responsible for the novel nucleoside triphosphate pyrophosphohydrolase activity shared by the two enzymes, and by another protein of unknown function, the product of the E. coli orf17 gene (Takahagi, M., Iwasaki, H., Nakata, A., and Shinegawa, H. (1991) J. Bacteriol. 173, 5747-5753).

Amino Acid Sequence↗
PubMed1994

Dual divalent cation requirement of the MutT dGTPase. Kinetic and magnetic resonance studies of the metal and substrate complexes.

Kinetic analyses of both the Mn(2+)- and Mg(2+)-activated hydrolysis of dGTP by MutT show the requirement for two divalent cations. Whereas Mn2+ supports a 20-fold lower kcat (0.19 s-1) than Mg2+ (4.0 s-1), the Km of Mn2+.dGTP (6.3 microM) is 45-fold lower than that of Mg2+.dGTP (284 microM). Adenosine 5'-(alpha,beta-methylenetriphosphate) (AMPCPP) is a linear competitive inhibitor with respect to dGTP with a Ki for Mg2+.AMPCPP (42 microM) which is 57-fold lower than the Ki of Mg2+.AMPCPP (2.4 mM). Such tightening suggests that a metal-bridge E.M2+.NTP.M2+ complex is the catalytically active species. The 12 dissociation constants describing the quaternary MutT.M2+.AMPCPP.M2+ complex were evaluated for both Mn2+ and Mg2+, using EPR and NMR methods. MutT binds a single Mn2+ with a Kd of 130 +/- 40 microM in reasonable agreement with the kinetically determined activator constant of Mn2+ of 230 +/- 72 microM. The MutT.AMPCPP complex binds two Mn2+ ions, the weaker of which has a Kd of 16 +/- 2 microM in agreement with the kinetically determined KmMn2+ of 26 +/- 10 microM. MutT.Mn2+ binds Mn2+.AMPCPP with Kd of 16 +/- 4 microM, whereas MutT alone binds Mn2+.AMPCPP with a Kd of 135 +/- 30 microM. The 17-fold enhanced paramagnetic effect of Mn2+ on the longitudinal relaxation rate of water protons found with the binary MutT.Mn2+ complex decreases to 4.7-fold upon binding of AMPCPP and to 8.7-fold upon binding of Mn2+.AMPCPP, further supporting a metal-bridge MutT.M2+.NTP.M2+ complex. By competition with Mn2+ MutT binds Mg2+ at one site with a Kd of 7.5 mM, and MutT.AMPCPP binds Mg2+ at two sites, the weaker of which has a Kd of 0.9 mM. These values are comparable to the kinetically determined KaMg of 15 +/- 7 mM and KmMg of 1.7 +/- 0.7 mM, respectively. Studies with the racemic, substitution-inert beta, gamma-bidentate tetraamminecobalt (III)-beta,gamma-phosphate-ATP (Co3+(NH3)4ATP) complex show that MutT slowly hydrolyzes only the lambda stereoisomer but requires Mg2+ or Mn2+ to do so, confirming a dual metal ion requirement.

Adenosine Triphosphate↗
PubMed1994

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 pyrophosphohydrolases.

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 co-ordinately regulated. Nucleotide sequence determination reveals that the mutX gene encodes a 17,870 Da protein (154 residues) which exhibits significant homology with the MutT protein of Escherichia coli, a nucleoside triphosphatase (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.

Amino Acid Sequence↗
PubMed1993

Sequence-specific assignments of the backbone 1H, 13C, and 15N resonances of the MutT enzyme by heteronuclear multidimensional NMR.

The MutT protein, a 129-residue enzyme from Escherichia coli which prevents A.T-->C.G mutations, catalyzes the hydrolysis of nucleoside triphosphates (NTP) to nucleoside monophosphates (NMP) and pyrophosphate [Bhatnagar, S. K., Bullions, L. C., & Bessman, M. J. (1991) J. Biol. Chem. 266, 9050-9054], by a mechanism involving nucleophilic substitution at the rarely attacked beta-phosphorus of NTP [Weber, D. J., Bhatnagar, S. K., Bullions, L. C., Bessman, M. J., & Mildvan, A. S. (1992a) J. Biol. Chem. 267, 16939-16942]. The bacterial MutT gene was inserted into the plasmid pET-11b under control of the T7 promoter and overexpressed in minimal media, permitting labeling of MutT with 13C and/or 15N. The yield after purification of the soluble fraction was approximately 35 mg of homogeneous MutT/L with physical and enzymatic properties indistinguishable from those of the originally isolated enzyme. Essentially complete sequence-specific assignments of the backbone HN, N, C alpha, H alpha, and CO resonances of the free enzyme (1.5 mM) were made at pH 7.4 and 32 degrees C, by heteronuclear double- and triple-resonance experiments using a modified Bruker AM 600 NMR spectrometer. Specifically, 1H[15N]HSQC, 1H[15N]TOCSY-HMQC, and 1H[15N]NOESY-HMQC experiments were done with uniformly 15N-labeled enzyme. A 1H[15N] HSQC experiment was done with selective [alpha-15N]Lys-labeled enzyme. Also HNCA, HN(CO)CA, HNCO, constant time 1H[13C]HSQC, HCACO, and HCA(CO)N experiments were done with uniformly 13C- and 15N-labeled enzyme. Sequence-specific assignments were initiated from HN and 15N chemical shifts of Gly residues and of selectively labeled Lys residues in 1H[15N]HSQC experiments. They were confirmed by C alpha chemical shifts of Ala residues uniquely identified by residual coupling to C beta resonances in constant time 1H[13C]HSQC experiments. The sequence-specific assignments proceeded bidirectionally, terminating at Pro residues and at residues with undetectable NH signals, and the segments were linked to complete the backbone assignments. The backbone assignments reported here have permitted the interpretation of NOEs in the elucidation of the solution secondary structure of MutT, and the C alpha and H alpha chemical shifts have provided an independent approach to identifying secondary structural elements and to define their extent [Weber, D. J., Abeygunawardana, C., Bessman, M. J., & Mildvan, A. S. (1993) Biochemistry (following paper in this issue)].

Amino Acid Sequence↗
PubMed1993

Secondary structure of the MutT enzyme as determined by NMR.

The MutT enzyme (129 amino acids) catalyzes the hydrolysis of nucleoside triphosphates (NTP) to nucleotides (NMP) and pyrophosphate by nucleophilic substitution at the rarely attacked beta-phosphorus of NTP [Weber, D. J., Bhatnagar, S. K., Bullions, L. L., Bessman, M. J., & Mildvan, A. S. (1992) J. Biol. Chem. 267, 16939-16942]. Backbone NMR assignments for the H alpha, 13C alpha, HN, 15N, and carbonyl 13C' resonances, based on heteronuclear methods have been reported for MutT [Abeygunawardana, C., Weber, D. J., Frick, D. N. Bessman, M. J., & Mildvan, A. S. (1993) Biochemistry (preceding paper in this issue)]. Here, we report the secondary structure of MutT in solution on the basis of these assignments, NOE data derived from 2D and 3D homonuclear and heteronuclear NMR spectra, and amide NH exchange data. Consistent with near neighbor NOEs, H alpha and C alpha chemical shifts, and amide exchange rates, MutT contains two alpha-helices spanning residues 47-59 (helix 1) and residues 119-128 (helix 2), respectively. The helical content predicted from NMR (17.8 +/- 1.0%) is consistent with that predicted by circular dichroism spectroscopy (20.9 +/- 5.4%). A mixed parallel and antiparallel beta-sheet with five beta-strands (A-E) consists of residues A, 3-13; B, 18-24; C, 70-74; D, 79-87; and E, 102-106.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗
PubMed1992

NMR and isotopic exchange studies of the site of bond cleavage in the MutT reaction.

The MutT protein, which prevents AT----CG transversions during DNA replication, hydrolyzes nucleoside triphosphates to yield nucleoside monophosphates and pyrophosphate. The hydrolysis of dGTP by the MutT protein in H(2)18O-enriched water, when monitored by high resolution 31P NMR spectroscopy at 242.9 MHz, showed 18O labeling of the pyrophosphate product, as manifested by a 0.010 +/- 0.002 ppm upfield shift of the pyrophosphate resonance, and no labeling of the dGMP product. This establishes that the reaction proceeds via a nucleophilic substitution at the beta-phosphorus of dGTP with displacement of dGMP as the leaving group. No exchange of 32P-labeled dGMP into dGTP was detected, indicating that water attacks dGTP directly or, less likely, an irreversibly formed pyrophosphoryl-enzyme intermediate. No exchange of 32P-labeled pyrophosphate into dGTP was observed, consistent with nucleophilic substitution at the beta-phosphorus of dGTP. Only six enzymes, all synthetases, have previously been shown to catalyze nucleophilic substitution at the beta-phosphorus of nucleoside triphosphate substrates. The MutT protein is the first hydrolase shown to do so.

Bacterial Proteins↗
PubMed1991

Characterization of the mutT nucleoside triphosphatase of Escherichia coli.

The mutT protein, which prevents A:T----C:G transversions during DNA replication, has the following enzymatic properties. Although it prefers dGTP as a substrate, it hydrolyzes all of the canonical nucleoside triphosphates to some extent. It has no activity in the absence of divalent cations, is maximally activated by magnesium ions, and has a pH optimum of 9.0. Nucleoside triphosphates are hydrolyzed according to the following equation. dGTP----dGMP + PPi Studies with nucleotide analogues suggest that the enzyme may prefer the syn rather than the anti conformation of the nucleoside triphosphates, which might explain the role it plays in preventing mutations.

Base Sequence↗
PubMed1991

Crystallization and preliminary X-ray diffraction studies on the mutT nucleoside triphosphate pyrophosphohydrolase of Escherichia coli.

The mutT nucleoside triphosphatase, which prevents AT----CG transversions during DNA replication, has been crystallized from ammonium sulfate utilizing a novel technique involving vapor diffusion in capillaries. X-ray diffraction analysis has revealed that the crystals are monoclinic, space group P2(1), with cell constants a = 34.14, b = 72.54, c = 56.38, and beta = 98.90. The Vm value of 2.31 A3/Da is consistent with two molecules of enzyme per asymmetric unit. The crystals are reasonably stable in the x-ray beam, and a data set to 2.5 A resolution has been collected for native protein. There is evidence that the crystals diffract to at least 2.1 A.

Crystallography↗
PubMed1991

dGTP triphosphohydrolase, a unique enzyme confined to members of the family Enterobacteriaceae.

The enzyme dGTP triphosphohydrolase (dGTPase; EC 3.1.5.1) was assayed in partially purified extracts of several genera of bacteria, and it was found to be strictly confined to members of the family Enterobacteriaceae. Whereas 11 of 12 enteric bacteria had comparable activity for this enzyme, 8 of 8 nonenteric bacteria, including species in the very closely related genera Vibrio and Aeromonas, did not assay positively for this enzyme. When challenged with Escherichia coli anti-dGTPase antiserum, the active enzymes fell into three groups, retaining 0, approximately 50, or 100% of their original activity. A computer search has revealed an amino acid sequence in the E. coli enzyme which matches well with the single-stranded-DNA binding motif of Prasad and Chiu (J. Mol. Biol. 193:579-584, 1987) and may account for the enzyme's observed interaction with DNA. As far as we are aware, this is the only enzymatic activity so far reported to be present solely in the enteric bacteria.

Amino Acid Sequence↗
PubMed1990

Primary structure of the deoxyguanosine triphosphate triphosphohydrolase-encoding gene (dgt) of Escherichia coli.

The complete nucleotide sequence has been determined for a 2027-bp region that encompasses the structural gene (dgt) encoding deoxyguanosine triphosphate triphosphohydrolase (dGTPase) from Escherichia coli. The gene resides between the htrA and dapD loci at 3.75-3.8' on the bacterial chromosome. Using homologous recombination in a recD recipient, a dgt- bacterial strain was constructed that was deficient in producing functional dGTPase. Comparison of dGTP pools in this and other strains revealed that dGTPase synthesized in vivo does to some degree modulate the level of dGTP in the bacterial cell, yet the magnitude of this modulation may be insufficient to explain the physiological function of dGTPase.

Amino Acid Sequence↗
PubMed1990

Bacteriophage T4 deoxynucleotide kinase: gene cloning and enzyme purification.

Gene 1 of bacteriophage T4 has been cloned into a lambda pL expression vector, resulting in the overproduction of deoxynucleotide kinase. A procedure that includes affinity chromatography on Cibacron Blue F3GA-agarose has been used to purify milligram quantities of enzymes from a 2-liter culture. The enzyme has been partially characterized in vitro and in vivo, and it appears to be identical to the deoxynucleotide kinase isolated from T4-infected Escherichia coli. These results prove the earlier contention that the phosphorylation of three dissimilar deoxynucleotides (5-hydroxymethyldeoxycytidylate, dTMP, and dGMP), to the exclusion of most others, is catalyzed by a single protein.

Amino Acid Sequence↗
PubMed1989

Location and molecular cloning of the structural gene for the deoxyguanosine triphosphate triphosphohydrolase of Escherichia coli.

The structural gene for deoxyguanosine triphosphate triphosphohydrolase (dGTPase) (EC 3.1.5.1) and its regulator, optA, have been located on a lambda phage carrying a 17.5kb Escherichia coli DNA insert. The DNA fragment has been excised and ligated into pBR325 and also transferred to another lambda vector. From the results of transduction and transformation experiments, we find that the structural gene for dGTPase is very closely linked to optA and dapD, which locates it at approximately 3.6 minutes on the genetic map of E. coli K12. We propose the mnemonic dgt as the designation for the structural gene for this enzyme.

Bacteriophage lambda↗