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Ja, Cowan

doi:10.1023/a:1016022730880

Cited by 369 publications

(168 citation statements)

References 28 publications

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“…FMP) are not activated by Mg 2ϩ and that the Mg 2ϩ -dependent pK a is consistent with the deprotonation of diphosphate (21). Additionally, this is a common mode of activation by magnesium for enzyme-catalyzed reactions of diphosphate substrates (30). On the other hand, the manganese affinity for the FTase ternary complex is only 1.8-fold tighter than magnesium, whereas manganese binds to diphosphate of ADP with a 6-fold tighter affinity than magnesium (45).…”

Section: Figmentioning

confidence: 73%

“…In the crystal structure of an inactive FTase ternary complex with bound manganese, the only manganese ligands observed were the two oxygens of the diphosphate-like moiety of the FPP analog (26). In most enzymes that use a catalytic magnesium ion, there is at least one carboxylate ligand from the enzyme that coordinates the magnesium ion in the active site (29,30). In the class I terpenoid synthase enzymes, which catalyze a prenyl transfer reaction, there is an aspartate-rich region of the enzyme active site, often a DDXXD motif, that binds the magnesium ion (31)(32)(33).…”

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confidence: 99%

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Mutagenesis Studies of Protein Farnesyltransferase Implicate Aspartate β352 as a Magnesium Ligand

Pickett

Bowers

Fierke

2003

Journal of Biological Chemistry

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Protein farnesyltransferase (FTase) 1 catalyzes the transfer of a 15-carbon farnesyl chain from farnesyl diphosphate (FPP) to a C-terminal cysteine sulfur of protein substrates in the cell (1). Protein or peptide substrates of FTase contain a C-terminal CaaX sequence, whereas C represents the cysteine that becomes farnesylated, a 1 and a 2 are typically small aliphatic amino acids, and X is commonly methionine, serine, glutamine, or alanine (2). FTase is known to modify several important proteins in the cell cycle machinery, including Ras, Rho B, CENP-E, and CENP-F (3). Farnesylation of proteins enhances membrane association and specific protein-protein interactions and therefore is thought to be important for localization of proteins in the cell (4 -6). After farnesylation by FTase, proteins are further processed by the CaaX protease Rce1 and the methyltransferase Icmt, producing a Cterminal farnesylated cysteine methyl ester as the end product of this pathway (7,8). Protein farnesyltransferase inhibitors are currently being evaluated in clinical trials for the treatment of several types of cancer, including acute myeloid leukemia, metastatic breast cancer, and non-small cell lung cancer (9, 10).Farnesylation is just one type of protein prenylation; proteins can also be modified with geranylgeranyl groups in a reaction catalyzed by geranylgeranyl transferase type I or type II (GGTase I and GGTase II) (11, 12). GGTase I also recognizes protein substrates with a C-terminal CaaX motif, but GGTase I substrates most frequently contain a leucine or phenylalanine at the X position (13, 14). FTase and GGTase I are structurally homologous heterodimers with identical ␣ subunits and differing ␤ subunits (15). Prenyltransferases contain an active site zinc ion that is required for activity; FTase and GGTase II also use a magnesium ion to facilitate catalysis (16 -19). Surprisingly, GGTase I apparently catalyzes the same prenyl transfer reaction with no dependence on magnesium ions (20).In FTase, the magnesium ion has been proposed to coordinate to the diphosphate of FPP, but the exact location of the magnesium binding site has not yet been identified (21). The bound zinc ion of FTase coordinates to the cysteine sulfur of the protein substrate, lowering the pK a to form a zincthiolate at neutral pH (22, 23). In the proposed farnesylation transition state of FTase (Scheme 1), positive charge accumulates on the C-1 of FPP, and the diphosphate leaving group develops additional negative charge (24, 25). Bound magnesium is proposed to stabilize the negative charge buildup on the diphosphate moiety and activate the leaving group (21). Although magnesium is not absolutely required for FTase catalysis, millimolar concentrations of magnesium ions enhance the rate constant for product formation by 700-fold (24).Several pieces of experimental evidence indicate that the diphosphate of FPP forms part of the magnesium binding site. X-ray crystallography of the FTase ternary complex in the presence of manganese showed a manganese ion bo...

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Section: Figmentioning

confidence: 73%

mentioning

confidence: 99%

Mutagenesis Studies of Protein Farnesyltransferase Implicate Aspartate β352 as a Magnesium Ligand

Pickett

Bowers

Fierke

2003

Journal of Biological Chemistry

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“…In mutational studies (29), Lys-87 was identified as an important contact residue for DNA binding, as evidenced by a 50% decrease in DNA binding affinity when alanine was substituted for Lys-87 in T4 RNase H. In the absence of an active site rearrangement, the general reaction mechanism for the FEN-1 family probably differs from that proposed for the Klenow fragment. Further studies will be required for a more comprehensive understanding of the mechanisms of action of this family of enzymes (24,(45)(46)(47).…”

Section: Discussionmentioning

confidence: 99%

Crystal Structure of Bacteriophage T4 5′ Nuclease in Complex with a Branched DNA Reveals How Flap Endonuclease-1 Family Nucleases Bind Their Substrates

Devos

Tomanicek

Jones

et al. 2007

Journal of Biological Chemistry

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Bacteriophage T4 RNase H, a flap endonuclease-1 family nuclease, removes RNA primers from lagging strand fragments. It has both 5 nuclease and flap endonuclease activities. Our previous structure of native T4 RNase H (PDB code 1TFR) revealed an active site composed of highly conserved Asp residues and two bound hydrated magnesium ions. Here, we report the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a flap endonuclease-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the helix-hairpin-helix motif class 2. The 5 arm crosses over the active site, extending below the bridge (helical arch) region. Cleavage assays of this DNA substrate identify a primary cut site 7-bases in from the 5 arm. The scissile phosphate, the first bond in the duplex DNA adjacent to the 5 arm, lies above a magnesium binding site. The less ordered 3 arm reaches toward the C and N termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp, respectively. In the crystal structure, the scissile bond is located within the double-stranded DNA, between the first two duplex nucleotides next to the 5 arm, and lies above a magnesium binding site. This complex provides important insight into substrate recognition and specificity of the flap endonuclease-1 enzymes.The flap endonuclease-1 (FEN-1) 3 nuclease family is conserved in sequence and structure from bacteriophage to humans. These nucleases play essential roles in DNA replication by removing the RNA primers from lagging strand fragments. In addition, FEN-1-related nucleases are important in long-patch base excision repair and in maintenance of genomic stability. Homozygous knockouts of FEN-1 in mice are lethal to embryos (for review, see Refs. 1-3).Like other family members, bacteriophage T4-encoded RNase H shows 5Ј to 3Ј exonuclease activity on either RNA/ DNA or DNA/DNA duplexes and endonuclease activity on either flap or fork DNA structures (4, 5). T4 rnh deletion mutants give no phage production and accumulate unligated, lagging strand fragments in Escherichia coli hosts with defective polymerase I 5Ј nuclease (6). In addition, the mutants are hypersensitive to UV irradiation and anti-tumor agents (7).FEN-1 family nuclease activities are modulated by interactions with DNA replication clamps (e.g. eukaryotic proliferating cell nuclear antigen and T4 gene 45 clamp) and single-stranded DNA-binding proteins (e.g. eukaryotic replication protein A and T4 gene 32 protein) (5, 8 -12). Human FEN-1 nuclease is also stimulated by interactions with the Werner (13, 14) and Bloom (15) syndrome helicases as well as the Rad9-Rad1-Hus1 (9-1-1) checkpoint clamp (16).T4 RNase H 5Ј nuclease removes a short oligonucleotide (1-4 bases) each time it binds its substrate. T4 32 protein, binding on single-stranded DNA behind the nuclease, increases its processivity so that about 10 -50 short oligonucleotides are removed in a single binding event (5). On nicked sub...

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“…Furthermore, Mg 2ϩ stabilizes the codon-anticodon interaction in the A site and influences the binding of ribosome recycling factor (RRF) to the ribosome (31-34). In addition to its involvement in the ribosome, Mg 2ϩ has a crucial role in numerous biological processes and cellular functions, such as the activation and catalytic reactions of hundreds of enzymes, utilization of ATP, and maintenance of genomic stability (35,36).…”

mentioning

confidence: 99%

Defect in the Formation of 70S Ribosomes Caused by Lack of Ribosomal Protein L34 Can Be Suppressed by Magnesium

et al. 2014

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dTo elucidate the biological functions of the ribosomal protein L34, which is encoded by the rpmH gene, the rpmH deletion mutant of Bacillus subtilis and two suppressor mutants were characterized. Although the ⌬rpmH mutant exhibited a severe slowgrowth phenotype, additional mutations in the yhdP or mgtE gene restored the growth rate of the ⌬rpmH strain. Either the disruption of yhdP, which is thought to be involved in the efflux of Mg 2؉ , or overexpression of mgtE, which plays a major role in the import of Mg 2؉ , could suppress defects in both the formation of the 70S ribosome and growth caused by the absence of L34. Interestingly, the Mg 2؉ content was lower in the ⌬rpmH cells than in the wild type, and the Mg 2؉ content in the ⌬rpmH cells was restored by either the disruption of yhdP or overexpression of mgtE. In vitro experiments on subunit association demonstrated that 50S subunits that lacked L34 could form 70S ribosomes only at a high concentration of Mg 2؉ . These results showed that L34 is required for efficient 70S ribosome formation and that L34 function can be restored partially by Mg 2؉ . In addition, the Mg 2؉ content was consistently lower in mutants that contained significantly reduced amounts of the 70S ribosome, such as the ⌬rplA (L1) and ⌬rplW (L23) strains and mutant strains with a reduced number of copies of the rrn operon. Thus, the results indicated that the cellular Mg 2؉ content is influenced by the amount of 70S ribosomes.T he eubacterial ribosome (70S), which plays a central role in protein synthesis, is composed of a small (30S) subunit and a large (50S) subunit. The small subunit is comprised of the 16S rRNA and more than 20 proteins, whereas the large subunit is comprised of the 23S and 5S rRNAs and more than 30 proteins (1, 2). The molecular mechanisms of translation have been elucidated in detail by the convergence of various approaches, including crystal structure analysis (3-8). The individual functions of several ribosomal proteins have also been elucidated by biochemical and genetic analyses, including reconstitution and mutational analysis. For example, ribosomal protein L2 plays important roles in the assembly of the ribosomal subunits, binding of the tRNA to the A and P sites, peptidyltransferase activity, and formation of the peptide bond (9-13). However, in general, disruption of the genes that encode the ribosomal proteins has been avoided as a means of identifying protein function, because these genes, which are highly conserved in bacteria, have been considered essential for cell proliferation (14). Nonetheless, recently, it was found that 22 of the 54 Escherichia coli genes for ribosomal proteins could be deleted on an individual basis (15,16). We have also shown that out of the 57 ribosomal-protein-encoding genes that have been annotated in the Gram-positive bacterium Bacillus subtilis, at least 22 genes are not individually essential for cell proliferation (17). The rpmH gene encoding ribosomal protein L34, which is a component of the large subunit, is one of the...

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Untitled

Cited by 369 publications

References 28 publications

Mutagenesis Studies of Protein Farnesyltransferase Implicate Aspartate β352 as a Magnesium Ligand

Mutagenesis Studies of Protein Farnesyltransferase Implicate Aspartate β352 as a Magnesium Ligand

Crystal Structure of Bacteriophage T4 5′ Nuclease in Complex with a Branched DNA Reveals How Flap Endonuclease-1 Family Nucleases Bind Their Substrates

Defect in the Formation of 70S Ribosomes Caused by Lack of Ribosomal Protein L34 Can Be Suppressed by Magnesium

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