All three residues of the Tn 10 transposase DDE catalytic triad function in divalent metal ion binding 1 1Edited by M. Gottesman

Allingham, John S.; Pribil, Patrick; Haniford, David B.

doi:10.1006/jmbi.1999.2837

Cited by 50 publications

(33 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cysteine mutagenesis studies of Tn10 transposase (29) demonstrated that all three DDE residues function in divalent metal binding and that the glutamate residue has a role in target capture. All IN DDE mutants display reduced 3Ј end processing, strand transfer, and disintegration, and mutation of any of these residues in related transposases and polynucleotidyltransferases diminishes their respective activities.…”

Section: Discussionmentioning

confidence: 99%

Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

Grobler

Stillmock²,

Hu³

et al. 2002

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

379

325

View full text Add to dashboard Cite

The process of integrating the reverse-transcribed HIV-1 DNA into the host chromosomal DNA is catalyzed by the virally encoded enzyme integrase (IN). Integration requires two metal-dependent reactions, 3 end processing and strand transfer. Compounds that contain a diketo acid moiety have been shown to selectively inhibit the strand transfer reaction of IN in vitro and in infected cells and are effective as inhibitors of HIV-1 replication. To characterize the molecular basis of inhibition, we used functional assays and binding assays to evaluate a series of structurally related analogs. These studies focused on investigating the role of the conserved carboxylate and metal binding. We demonstrate that an acidic moiety such as a carboxylate or isosteric heterocycle is not required for binding to the enzyme complex but is essential for inhibition and confers distinct metaldependent properties on the inhibitor. Binding requires divalent metal and resistance is metal dependent with active site mutants displaying resistance only when the enzymes are evaluated in the context of Mg 2؉ . The mechanism of action of these inhibitors is therefore likely a consequence of the interaction between the acid moiety and metal ion(s) in the IN active site, resulting in a functional sequestration of the critical metal cofactor(s). These studies thus have implications for modeling active site inhibitors of IN, designing and evaluating analogs with improved efficacy, and identifying inhibitors of other metal-dependent phosphotransferases.A n essential step in HIV replication is the integration of the reverse-transcribed viral genome into host chromosomal DNA by the virally encoded integrase (IN) protein (1-3). Integration is required for efficient long terminal repeat-driven transcription of the provirus for the production of viral proteins and RNA progeny. IN represents an important chemotherapeutic target, as its inactivation, either by mutagenesis or inhibition, blocks productive infection by HIV-1 (4-7).Integration is carried out in the cell in a series of distinct steps (8-10). First, IN cleaves the two terminal nucleotides from each 3Ј end of the viral DNA. The 3Ј processing reaction is carried out concurrently with or soon after reverse transcription in the cytoplasm. In the second step, strand transfer, IN catalyzes staggered nicking of the target chromosomal DNA and joining of each 3Ј end of the viral DNA to the 5Ј ends of the host DNA. Strand transfer is temporally and spatially separated from 3Ј processing and occurs after transport of the preintegration complex from the cytoplasm into the nucleus.Divalent metals such as Mg 2ϩ or Mn 2ϩ are required for both 3Ј processing and strand transfer and for the assembly of IN onto specific viral donor DNA to form a complex competent to carry out either function (11)(12)(13) (18), L-731,988 and related DKAs inhibit integration and viral replication in cell culture (7). Mutations that confer resistance to DKAs have been identified and map to IN residues adjacent to D64 and E152. The in...

show abstract

Section: Discussionmentioning

confidence: 99%

Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

Grobler

Stillmock²,

Hu³

et al. 2002

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

379

325

View full text Add to dashboard Cite

show abstract

“…We note, however, that the AtpZ proteins of two alkaliphilic Bacillus species possess three contiguous acidic residues at their C terminus. A discontinuous DDE motif has been shown to function in divalent metal binding (40), and contiguous acidic residues have been associated with Ca 2ϩ binding and translocating proteins (34,41).…”

Section: Effects Of Atpz Atpi or Atpzi Expression On The Phenotypes Ofmentioning

confidence: 99%

A tenth atp gene and the conserved atpI gene of a Bacillus atp operon have a role in Mg ²⁺ uptake

Hicks

Wang

Wei

et al. 2003

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

View full text Add to dashboard Cite

The atp operon of alkaliphilic Bacillus pseudofirmus OF4, as in most prokaryotes, contains the eight structural genes for the F-ATPase (ATP synthase), which are preceded by an atpI gene that encodes a membrane protein of unknown function. A tenth gene, atpZ, has been found in this operon, which is upstream of and overlapping with atpI. Most Bacillus species, and some other bacteria, possess atpZ homologues. AtpZ is predicted to be a membrane protein with a hairpin topology, and was detected by Western analyses. Deletion of atpZ, atpI, or atpZI from B. pseudofirmus OF4 led to a requirement for a greatly increased concentration of Mg 2؉ for growth at pH 7.5. Either atpZ, atpI, or atpZI complemented the similar phenotype of a triple mutant of Salmonella typhimurium (MM281), which is deficient in Mg 2؉ uptake. atpZ and atpI, separately and together, increased the Mg 2؉ -sensitive 45 Ca 2؉ uptake by vesicles of an Escherichia coli mutant that is defective in Ca 2؉ and Na ؉ efflux. We hypothesize that AtpZ and AtpI, as homooligomers, and perhaps as heterooligomers, are Mg 2؉ transporter, Ca 2؉ transporter, or channel proteins. Such proteins could provide Mg 2؉ , which is required by ATP synthase, and also support charge compensation, when the enzyme is functioning in the hydrolytic direction; e.g., during cytoplasmic pH regulation. P rokaryotic atp operons encode the cell membrane F-type ATPase (ATP synthase) that couples the energy of an electrochemical H ϩ gradient (or sometimes Na ϩ ), to the synthesis of ATP, from ADP and P i . In the reverse reaction, the ATPase hydrolyzes ATP concomitant with H ϩ (or Na ϩ ) efflux, thereby contributing to cytoplasmic pH regulation and͞or generation of a transmembrane electrochemical gradient under fermentative conditions (1-4). Most atp operons, like that of Escherichia coli, contain the eight structural genes for the ATPase, atpBEFHAGDC, which are preceded by atpI (5). The Escherichia coli atpI is expressed, and its product associates with the membrane, as predicted from its deduced sequence (6-9). Whereas there is no demonstrated effect of AtpI on expression or assembly of the ATPase, an atpI deletion strain of E. coli has been reported to have a reduced growth yield (7). There is no function established for this "mysterious ninth gene" (10) that accounts for such an effect. We report here the finding of another gene, encoding a membrane protein, that is upstream of the atpI gene, and within the atp operon of alkaliphilic Bacillus pseudofirmus OF4. This gene, designated atpZ, was discovered during attempts to introduce site-directed changes in alkaliphilespecific motifs of the membrane-embedded F-ATPase subunits of B. pseudofirmus OF4 (11). A cassette introduced just upstream of the putative atp promoter abolished atp expression. This finding led us to reexamine the location of the atp operon promoter, to the inclusion of atpZ in the extended operon, and then to an exploration of the effects of deleting atpI as well as atpZ. The results suggest a cation translocation function ...

show abstract

“…This motif is believed to be conserved among all transposases and retroviral integrases. The DDE residues serve to coordinate divalent metal ions that are required for catalysis (11)(12)(13)(14). A previous study has suggested that Asp-97 and Glu-326 contribute to the coordination of one metal ion, whereas Asp-97 and Asp-188 coordinate a second ion (15).…”

Section: Tn5 Transposase (Tnp)mentioning

confidence: 99%

Tn5 Transposase Active Site Mutations Suggest Position of Donor Backbone DNA in Synaptic Complex

Peterson

Reznikoff

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

Tn5 transposase (Tnp), a 53.3-kDa protein, enables the movement of transposon Tn5 by a conservative mechanism. Within the context of a protein and DNA synaptic complex, a single Tnp molecule catalyzes four sequential DNA breaking and joining reactions at the end of a single transposon. The three amino acids of the DDE motif (Asp-97, Asp-188, and Glu-326), which are conserved among transposases and retroviral integrases, have been shown previously to be absolutely required for all catalytic steps. To probe the effect of active site geometry on the ability to form synaptic complexes and perform catalysis, single mutations at each position of the DDE motif were constructed. The aspartates were changed to glutamates, and the glutamate was changed to an aspartate. These mutants were studied by performing in vitro binding assays using short oligonucleotide substrates simulating the natural substrates for the synaptic complex formation and subsequent transposition steps. The results indicate that the aspartate to glutamate mutations restrict synaptic complex formation with substrates resembling the natural transposon prior to transferred strand nicking. This suggests a structural model in which the donor backbone DNA, prior to nicking, occupies the same space that is invaded by the longer side chains present in the aspartate to glutamate mutants. Additionally, catalytic assays support the previous proposal that the active site coordinates two divalent metal ions.

show abstract

All three residues of the Tn 10 transposase DDE catalytic triad function in divalent metal ion binding 1 1Edited by M. Gottesman

Cited by 50 publications

References 35 publications

Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

A tenth atp gene and the conserved atpI gene of a Bacillus atp operon have a role in Mg ²⁺ uptake

Tn5 Transposase Active Site Mutations Suggest Position of Donor Backbone DNA in Synaptic Complex

Contact Info

Product

Resources

About

All three residues of the Tn 10 transposase DDE catalytic triad function in divalent metal ion binding 1 1Edited by M. Gottesman

Cited by 50 publications

References 35 publications

Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

A tenth atp gene and the conserved atpI gene of a Bacillus atp operon have a role in Mg 2+ uptake

Tn5 Transposase Active Site Mutations Suggest Position of Donor Backbone DNA in Synaptic Complex

Contact Info

Product

Resources

About

A tenth atp gene and the conserved atpI gene of a Bacillus atp operon have a role in Mg ²⁺ uptake