Cobamides and ribonucleotide reduction. IX. Monomeric, allosteric enzyme with a single polypeptide chain. Ribonucleotide reductase of Lactobacillus leichmannii

Panagou, Democleia; Orr, Malcolm D.; Dunstone, J. R.; Blakley, Raymond L.

doi:10.1021/bi00762a025

Cited by 80 publications

(56 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Initial efforts focused on the pUC plasmid containing the 1.1-kb insert from the PCR. The first 60 bases read from this cloned insert encoded 20 aa (aa [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] of the N-terminal end of RTPR. The first 18 nt, as expected, were those that encoded the N-terminal PCR primer.…”

Section: Methodsmentioning

confidence: 99%

Cloning, sequencing, and expression of the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii.

Booker

Stubbe

1993

Proc. Natl. Acad. Sci. U.S.A.

102

View full text Add to dashboard Cite

Ribonucleoside-triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacilus kichmannUi, a monomeric adenosylcobalamin-requiring enzyme, catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. The gene for this enzyme has been cloned and sequenced. In contrast to expectations based on mechanistic considerations, there is no statisticafly signicant sequence homology with the Escherichia coli reductase that requires a dinuclear-iron center and tyrosyl radical cofactor. The RTPR has been overexpressed and purified to homogeneity, yielding 90 mg of protein from 2.5 g of bacteria. Initial characterization of the recombinant RTPR indicates that its properties are identical to those of the RTPR isolated from L. kichmannu.Ribonucleotide reductases are uniquely responsible for converting nucleotides to deoxynucleotides in vivo (1-4). In contrast to most enzymes that play essential roles in metabolism in both prokaryotes and eukaryotes, the reductases do not appear, at least superficially, to have been evolutionarily conserved. In Escherichia coli, mammals, and herpesviruses, the enzymes possess two subunits, Rl and R2, each of which is homodimeric (a2132). The R2 subunit possesses a dinuclear iron center-tyrosyl radical cofactor that is essential for nucleotide reduction. The Rl subunit contains the binding sites for the NDP substrates and dNTP allosteric effectors, and the cysteines that are oxidized concomitant with substrate reduction. The ribonucleoside-triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii, the structurally simplest of all known reductases, is a monomer and requires adenosylcobalamin (AdoCbl) as a cofactor (5).Despite these diverse cofactors and quarternary structures, evidence suggests that the E. coli and L. leichmannii enzymes exhibit common patterns ofregulation and chemical mechanisms of reduction. The similarities in the allosteric regulatory properties are evident from investigations of the requirement for specific dNTPs to affect the reduction of specific purine and pyrimidine nucleotides (2, 6, 7). The similarities in mechanism of reduction are evident from investigations employing isotopically labeled nucleotide substrates and from investigations of the mechanisms of inhibition by 2'-chloro-2'-deoxynucleotides (3). With respect to the mechanism of reduction, both enzymes are proposed to initiate catalysis by generation of a protein radical that abstracts the hydrogen atom from the 3' carbon of the nucleotide substrate (3). The difference between the two enzymes is most apparent when considering the manner in which the two distinctly different cofactors give rise to the protein radical. For the E. coli reductase, it is proposed that the tyrosyl radical on the R2 subunit generates by long-range electron transfer a protein radical on the Rl subunit, which initiates turnover of the nucleotide (1, 8). The L. leichmanniiThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby...

show abstract

Section: Methodsmentioning

confidence: 99%

Cloning, sequencing, and expression of the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii.

Booker

Stubbe

1993

Proc. Natl. Acad. Sci. U.S.A.

102

View full text Add to dashboard Cite

show abstract

“…The small β chain contains an oxygen-linked diferric center and, in its active form, a stable tyrosyl free radical. Class II enzymes have a simpler structure (α or sometimes α 2 ) (38,39). Class III enzymes are α 2 β 2 heterotetramers, similar to class I (12).…”

Section: Three Classes: An Overviewmentioning

confidence: 99%

Ribonucleotide Reductases

Jordan

Reichard

1998

Annu. Rev. Biochem.

670

639

View full text Add to dashboard Cite

Ribonucleotide reductases provide the building blocks for DNA replication in all living cells. Three different classes of enzymes use protein free radicals to activate the substrate. Aerobic class I enzymes generate a tyrosyl radical with an ironoxygen center and dioxygen, class II enzymes employ adenosylcobalamin, and the anaerobic class III enzymes generate a glycyl radical from S-adenosylmethionine and an iron-sulfur cluster. The X-ray structure of the class I Escherichia coli enzyme, including forms that bind substrate and allosteric effectors, confirms previous models of catalytic and allosteric mechanisms. This structure suggests considerable mobility of the protein during catalysis and, together with experiments involving site-directed mutants, suggests a mechanism for radical transfer from one subunit to the other. Despite large differences between the classes, common catalytic and allosteric mechanisms, as well as retention of critical residues in the protein sequence, suggest a similar tertiary structure and a common origin during evolution. One puzzling aspect is that some organisms contain the genes for several different reductases. CONTENTS

show abstract

“…Most proteins which contain allosteric sites and which have been studied to date are multisubunit proteins. Only one other case in which an enzyme composed of only one polypeptide chain containing both an active site and a regulatory site has been investigated in detail [4]. Interest in such a system stems from the strong possibility that, in a monosubunit enzyme, all the structural changes occurring upon effector binding will be exclusively involved in the modulation of the active site activity.…”

mentioning

confidence: 99%

The Allosteric Activation of Mammalian α‐Amylase by Chloride

Levitzki

Steer

1974

European Journal of Biochemistry

131

View full text Add to dashboard Cite

&-Amylase from hog pancreas is shown to possess one binding site for C1-per molecule of enzyme with a dissociation constant of 3 x a t 25 "C. The chloride anion functions as an activating effector increasing kcat 30-fold towards either starch or p-nitrophenylmaltoside. No change in K , for either substrate is seen. Thc other monovalent anions Br-, I-, NO,-, NO,-, C104-, SCN-, N,-and CNO-can substitute for chloride but their effect on krat decreases as their anionic radius increases. Chloride binding induces a subtle con€ormational change in the enzyme reflected by the suppression of the exchange of 26 protons and a 240-fold increase in the amylase-Ca2+ binding constant from 8.3 x 108 to 2 x loll M-I. The conformational change is minor since it is not detected by circular dichroism, protein fluorescence or by specific probes attached to the enzyme. The ability of small structural changes to induce large catalytic acceleration is discussed.It has been shown that effector ligands can regulate the catalytic power of active sites of enzyme [1,2]. Enzymes can be switched on and off via the binding of ligands to sites not overlapping with the active site. The modulation of the active site activity by ligands dissimilar in structure to either the substrate or the products has often been classified as an allosteric eiyect [3]. Allosteric effects do not necessarily involve cooperative effects in the binding of the substrate or the modulating ligand. The modulating effector ligand is often called the allosteric ligand and the binding site for the latter, the allosteric site on the regulatory site. Most proteins which contain allosteric sites and which have been studied to date are multisubunit proteins. Only one other case in which an enzyme composed of only one polypeptide chain containing both an active site and a regulatory site has been investigated in detail [4]. Interest in such a system stems from the strong possibility that, in a monosubunit enzyme, all the structural changes occurring upon effector binding will be exclusively involved in the modulation of the active site activity. On the other hand, in a niultisubunit enzyme, structural changes in the quaternary structure are always involved and may not be directly related to the active site activity.Thc pancreatic n-amylase (50000 molecular weights) is historically the first enzyme which was Abbreaiations. Nbs,, 5,B'-dithio-bis(2-nitrobenzoic acid) ;Enzyme. %-Amylase or a-1,4-glucano hydrolaso (EC Sbs, 5-thio-2-nitrobenzoate; CD, circular dichroism. 3.2.1.1).found to be modulated by an effector ligand [5].The physiological effector ligand is C1-which causes a strong activation of the n-amylolytic activity of pancreatic and salivary n-amylase [S]. Although this effect has been known for over 90 years [7], very few detailed reports dealing with the chloride activation have appeared [5,6,S]. I n this communication an attempt is made to investigate the molecular basis by which C1-activates this monomeric enzyme. EXPERIMENTAL PROCEDURE MaterialsAll chemicals used were...

show abstract

Cobamides and ribonucleotide reduction. IX. Monomeric, allosteric enzyme with a single polypeptide chain. Ribonucleotide reductase of Lactobacillus leichmannii

Cited by 80 publications

References 64 publications

Cloning, sequencing, and expression of the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii.

Cloning, sequencing, and expression of the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii.

Ribonucleotide Reductases

The Allosteric Activation of Mammalian α‐Amylase by Chloride

Contact Info

Product

Resources

About