title: Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis.
abstract: Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced β-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5'-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg(2+) or Mn(2+) ions. 5'-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 Å, revealing a 'doughnut-shaped' toroidal trimeric arrangement with a central tapered channel, analogous to that of λ-exonuclease (Exo) from bacteriophage-λ. Active sites containing two bound Mg(2+) ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases.
authors: State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071, China.
authors: Wen
authors: W
authors: Yang
authors: Wen-yang
authors: WY
authors: Chen
authors: Hui
authors: H
authors: Wang
authors: John W S
authors: JW
authors: Ho
authors: Jian-Dong
authors: JD
authors: Huang
authors: Patrick C Y
authors: PC
authors: Woo
authors: Susanna K P
authors: SK
authors: Lau
authors: Kwok-Yung
authors: KY
authors: Yuen
authors: Qionglin
authors: Q
authors: Zhang
authors: Weihong
authors: W
authors: Zhou
authors: Mark
authors: M
authors: Bartlam
authors: Rory M
authors: RM
authors: Watt
authors: Zihe
authors: Z
authors: Rao
mesh_terms: D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D009346:Neisseriaceae / Q000201:enzymology*; D010766:Phosphorylation; D055503:Protein Multimerization
chemical_list: D004277:DNA, Single-Stranded; D008670:Metals; D004247:DNA; D005090:Exodeoxyribonucleases