Identification of the amino acid residues involved in an active site of Escherichia coli ribonuclease H by site-directed mutagenesis.

Kanaya, Shigenori; Kohara, Atuko; Miura, Yasühiro; Sekiguchi, Akira; Iwai, Shigenori; Inoue, Hideo; Ohtsuka, Eiko; Ikehara, Morio

doi:10.1016/s0021-9258(19)39607-3

Cited by 172 publications

(59 citation statements)

References 50 publications

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“…RNase H effectively catalyses the selective cleavage of the phosphodiester bonds in the RNA moiety of the DNA:RNA double helix in the presence of divalent cations (preferentially Mg 2+ ) to generate products with 5 0 -phosphate and 3 0 -hydroxy termini. 1,2 Among the various known RNase H enzymes, E. coli RNase HI has been most extensively studied for structurefunction relationships by X-ray analysis, [3][4][5] site-directed mutagenesis, 6 and NMR studies. 7,8 The crystal structure of E. coli RNase HI was first determined in 1990 and revealed a novel a/b fold containing four carboxylate residues (Asp10, Glu48, Asp70, Asp134) in the catalytic center.…”

Section: Introductionmentioning

confidence: 99%

Atomistic details of the associative phosphodiester cleavage in human ribonuclease H

Elsässer

Fels

2010

Phys. Chem. Chem. Phys.

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During translation of the genetic information of DNA into proteins, mRNA is synthesized by RNA polymerase and after the transcription process degraded by RNase H. The endoribonuclease RNase H is a member of the nucleotidyl-transferase (NT) superfamily and is known to hydrolyze the phosphodiester bonds of RNA which is hybridized to DNA. Retroviral RNase H is part of the viral reverse transcriptase enzyme that is indispensable for the proliferation of retroviruses, such as HIV. Inhibitors of this enzyme could therefore provide new drugs against diseases like AIDS. In our study we investigated the molecular mechanism of RNA cleavage by human RNase H using a comprehensive high level DFT/B3LYP QM/MM theoretical method for the calculation of the stationary points and nudged elastic band (NEB) and free energy calculations to identify the transition state structures, the rate limiting step and the reaction barrier. Our calculations reveal that the catalytic mechanism proceeds in two steps and that the nature of the nucleophile is a water molecule. In the first step, the water attack on the scissile phosphorous is followed by a proton transfer from the water to the O2P oxygen and a trigonal bipyramidal pentacoordinated phosphorane is formed. Subsequently, in the second step the proton is shuttled to the O3' oxygen to generate the product state. During the reaction mechanism two Mg(2+) ions support the formation of a stable associated in-line S(N)2-type phosphorane intermediate. Our calculated energy barrier of 19.3 kcal mol(-1) is in excellent agreement with experimental findings (20.5 kcal mol(-1)). These results may contribute to the clarification and understanding of the RNase H reaction mechanism and of further enzymes from the RNase family.

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Section: Introductionmentioning

confidence: 99%

Atomistic details of the associative phosphodiester cleavage in human ribonuclease H

Elsässer

Fels

2010

Phys. Chem. Chem. Phys.

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“…This histidine has been reported to be significantly involved in proton transfer during the nucleophilic attack in the cleavage reaction (Oda et al, 1993) and also to possibly be related to product release of RNase HI (Nowotny et al, 2007). Consistently, an H124A mutation leads to a substantial loss of the nuclease activity (Kanaya et al, 1990). Although 1 H NMR analyses have suggested an indirect interaction between His124 and the DEDD motif (Oda et al, 1993), the exact role of His124 during catalysis remains unclear due to the lack of an atomic structure of RNase HI showing a well defined conformation of His124.…”

Section: Introductionmentioning

confidence: 80%

“…Besides the substrate and divalent cations, the negatively charged electrostatic field formed by the carboxyl side chains of Asp and Glu residues, usually referred to as the DEDD motif, is well known to be a key player in RNase HI function. Structural analyses found that the DEDD motif coordinates the divalent cation at the active site, acting as metal ligands (Katayanagi et al, 1992), and constitutes the pivotal scaffold for the catalytic reaction (Kanaya et al, 1990). In addition to the highly conserved DEDD motif, a histidine adjacent to the active site (His124 in E. coli RNase HI) in the loop structure between 5 and 5 (Fig.…”

Section: Introductionmentioning

confidence: 99%

Pivotal role of a conserved histidine in Escherichia coli ribonuclease HI as proposed by X-ray crystallography

Liao¹,

Oyama²,

Kitagawa³

et al. 2022

Acta Crystallogr D Struct Biol

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The ribonuclease (RNase) H family of enzymes catalyze the specific cleavage of RNA strands of RNA/DNA hybrid duplexes and play an important role in DNA replication and repair. Since the first report of the crystal structure of RNase HI, its catalytic mechanisms, which require metal ions, have been discussed based on numerous structural and functional analyses, including X-ray crystallography. In contrast, the function of the conserved histidine residue (His124 in Escherichia coli) in the flexible loop around the active site remains poorly understood, although an important role was suggested by NMR analyses. Here, novel high-resolution X-ray crystal structures of E. coli RNase HI are described, with a particular focus on the interactions of divalent cations with His124 oriented towards the active site. The enzyme–Mg2+ complex contains two metal ions in the active site, one of which has previously been observed. The second ion lies alongside the first and binds to His124 in an octahedral coordination scheme. In the enzyme–Zn2+ complex a single metal ion was found to bind to the active site, showing a tetrahedral coordination geometry with the surrounding atoms, including His124. These results provide structural evidence that His124 plays a crucial role in the catalytic activity of RNase HI by interacting weakly and transiently with metal ions in the catalytic center.

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“…Doolittle et al [5] have found remarkable resemblances in amino acid sequences between RNase H from Eschericia coli (E. coli RNase H) and the RNase H domain of RTs from many retroviruses including Moloney murine leukemia virus (MuLV) and HIV-1. Recently, the tertiary structure of E. coli RNase H was revealed by two independent groups of Katayanagi et al [6] and Yang et al [7] Moreover, the active site of E. coli RNase H has been identified by protein engineering studies [8], and the substrate recognition mode of the enzyme has been studied by the present authors [9].…”

Section: Introductionmentioning

confidence: 81%

“…In the previous structural [6,7] and protein engineering [8] studies of E. coli RNase H, structurally and functionally essential residues have been revealed. Most of them are found to be well conserved in the amino acid sequences in RT-RNase H domains.…”

Section: Discussionmentioning

confidence: 99%

Structural models of ribonuclease H domains in reverse transcriptases from retroviruses

et al. 1991

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Tertiary models of ribonuclease H (RNase H) domains in reverse transcriptases (RTs) from Moloney murine leukemia virus (MuLV) and human immunodeficiency virus (HIV-1) were built based upon the X-ray structure of RNase H from Escherichia coli (E. coli RNase H). In two models of RT-RNase H domains, not only active site residues but also residues, which construct a hydrophobic core and hydrogen bonds, are located in the same positions as those of E. coli RNase H. The whole backbone structure and the electrostatic molecular surface of MuLV RT-RNase H model are similar to those of E. coli RNase H. On the contrary, HIV-1 RT-RNase H model lacks the third helix and the following loop, resulting no positive charge clusters around the hybrid recognition site. Referring the complex models of RTs with their substrate hybrid, the interaction between DNA-polymerase and RNase H domains in RTs was discussed.

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Identification of the amino acid residues involved in an active site of Escherichia coli ribonuclease H by site-directed mutagenesis.

Cited by 172 publications

References 50 publications

Atomistic details of the associative phosphodiester cleavage in human ribonuclease H

Atomistic details of the associative phosphodiester cleavage in human ribonuclease H

Pivotal role of a conserved histidine in Escherichia coli ribonuclease HI as proposed by X-ray crystallography

Structural models of ribonuclease H domains in reverse transcriptases from retroviruses

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