title: Structural and biochemical characterization of bacterial YpgQ protein reveals a metal-dependent nucleotide pyrophosphohydrolase.
abstract: The optimal balance of cellular nucleotides and the efficient elimination of non-canonical nucleotides are critical to avoiding erroneous mutation during DNA replication. One such mechanism involves the degradation of excessive or abnormal nucleotides by nucleotide-hydrolyzing enzymes. YpgQ contains the histidine-aspartate (HD) domain that is involved in the hydrolysis of nucleotides or nucleic acids, but the enzymatic activity and substrate specificity of YpgQ have never been characterized. Here, we unravel the catalytic activity and structural features of YpgQ to report the first Mn(2+)-dependent pyrophosphohydrolase that hydrolyzes (deoxy)ribonucleoside triphosphate [(d)NTP] to (deoxy)ribonucleoside monophosphate and pyrophosphate using the HD domain. YpgQ from Bacillus subtilis (bsYpgQ) displays a helical structure and assembles into a unique dimeric architecture that has not been observed in other HD domain-containing proteins. Each bsYpgQ monomer accommodates a metal ion and a nucleotide substrate in a cavity located between the N- and C-terminal lobes. The metal cofactor is coordinated by the canonical residues of the HD domain, namely, two histidine residues and two aspartate residues, and is positioned in close proximity to the β-phosphate group of the nucleotide, allowing us to propose a nucleophilic attack mechanism for the nucleotide hydrolysis reaction. YpgQ enzymes from other bacterial species also catalyze pyrophosphohydrolysis but exhibit different substrate specificity. Comparative structural and mutational studies demonstrated that residues outside the major substrate-binding site of bsYpgQ are responsible for the species-specific substrate preference. Taken together, our structural and biochemical analyses highlight the substrate-recognition mode and catalysis mechanism of YpgQ in pyrophosphohydrolysis.
authors.affiliation: Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, Chuncheon 200-701, Republic of Korea.
authors.forename: Ye Ji
authors.initials: YJ
authors.lastname: Jeon
authors.affiliation: Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, Chuncheon 200-701, Republic of Korea.
authors.forename: Sun Cheol
authors.initials: SC
authors.lastname: Park
authors.affiliation: Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, Chuncheon 200-701, Republic of Korea.
authors.forename: Wan Seok
authors.initials: WS
authors.lastname: Song
authors.affiliation: Lee Gil Ya Cancer and Diabetes Institute, College of Medicine, Gachon University, Incheon 406-840, Republic of Korea.
authors.forename: Ok-Hee
authors.initials: OH
authors.lastname: Kim
authors.affiliation: Lee Gil Ya Cancer and Diabetes Institute, College of Medicine, Gachon University, Incheon 406-840, Republic of Korea.
authors.forename: Byung-Chul
authors.initials: BC
authors.lastname: Oh
authors.affiliation: Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, Chuncheon 200-701, Republic of Korea; Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200-701, Republic of Korea. Electronic address: sungil@kangwon.ac.kr.
authors.forename: Sung-Il
authors.initials: SI
authors.lastname: Yoon
mesh_terms: D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003848:Deoxyguanine Nucleotides / Q000378:metabolism*; D007700:Kinetics; D008345:Manganese / Q000737:chemistry*; D008958:Models, Molecular; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D003848:Deoxyguanine Nucleotides; D011994:Recombinant Proteins; D008345:Manganese; C029603:deoxyguanosine triphosphate; D010744:Phosphoric Monoester Hydrolases
keywords: Crystal structure; HD domain; Manganese; Pyrophosphate; Substrate specificity