Biochemical and structural characterization of a DNA N6-adenine methyltransferase from Helicobacter pylori.
DNA N6-methyladenine modification plays an important role in regulating a variety of biological functions in bacteria. However, the mechanism of sequence-specific recognition in N6-methyladenine modification remains elusive. M1.HpyAVI, a DNA N6-adenine methyltransferase from Helicobacter pylori, shows more promiscuous substrate specificity than other enzymes. Here, we present the crystal structures of cofactor-free and AdoMet-bound structures of this enzyme, which were determined at resolutions of 3.0 Å and 3.1 Å, respectively. The core structure of M1.HpyAVI resembles the canonical AdoMet-dependent MTase fold, while the putative DNA binding regions considerably differ from those of the other MTases, which may account for the substrate promiscuity of this enzyme. Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity. Taken together, our data revealed the structural basis underlying DNA N6-adenine methyltransferase substrate promiscuity.
Department of Human Anatomy, Histology and Embryology, Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, and State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing, China.
Bo
B
Ma
Department of Human Anatomy, Histology and Embryology, Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, and State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing, China.
Ji
J
Ma
Institute of Immunology, The Third Military Medical University, Chongqing, China.
Dong
D
Liu
Institute of Immunology, The Third Military Medical University, Chongqing, China.
Ling
L
Guo
Institute of Immunology, The Third Military Medical University, Chongqing, China.
Huiling
H
Chen
Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
Jingjin
J
Ding
Institute of Immunology, The Third Military Medical University, Chongqing, China.
Wei
W
Liu
Department of Human Anatomy, Histology and Embryology, Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, and State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing, China.
Hongquan
H
Zhang
D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019175:DNA Methylation; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)
AdoMet-binding; DNA N6-adenine methyltransferase; Helicobacter pylori; Immune response; Immunity; Immunology and Microbiology Section; M1.HpyAVI; substrate recognition