title: Biochemical characterization of human SET and MYND domain-containing protein 2 methyltransferase.
abstract: SET and MYND domain-containing protein 2 (SMYD2) is a protein lysine methyltransferase that catalyzes the transfer of methyl groups from S-adenosylmethionine (AdoMet) to acceptor lysine residues on histones and other proteins. To understand the kinetic mechanism and the function of individual domains, human SMYD2 was overexpressed, purified, and characterized. Substrate specificity and product analysis studies established SMYD2 as a monomethyltransferase that prefers nonmethylated p53 peptide substrate. Steady-state kinetic and product inhibition studies showed that SMYD2 operates via a rapid equilibrium random Bi Bi mechanism at a rate of 0.048 ± 0.001 s(-1), with K(M)s for AdoMet and the p53 peptide of 0.031 ± 0.01 μM and 0.68 ± 0.22 μM, respectively. Metal analyses revealed that SMYD2 contains three tightly bound zinc ions that are important for maintaining the structural integrity and catalytic activity of SMYD2. Catalytic activity was also shown to be dependent on the GxG motif in the S-sequence of the split SET domain, as a G18A/G20A double mutant and a sequence deletion within the conserved motif impaired AdoMet binding and significantly decreased enzymatic activity. The functional importance of other SMYD2 domains including the MYND domain, the cysteine-rich post-SET domain, and the C-terminal domain (CTD), were also investigated. Taken together, these results demonstrated the functional importance of distinct domains in the SMYD family of proteins and further advanced our understanding of the catalytic mechanism of this family.
authors.affiliation: Oncology Innovative Medicines Unit, AstraZeneca Pharmaceuticals, 35 Gatehouse Drive, Waltham, Massachusetts 02451, USA. jiaquan.wu@astrazeneca.com
authors.forename: Jiaquan
authors.initials: J
authors.lastname: Wu
authors.forename: Tony
authors.initials: T
authors.lastname: Cheung
authors.forename: Christie
authors.initials: C
authors.lastname: Grande
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ferguson
authors.forename: Xiahui
authors.initials: X
authors.lastname: Zhu
authors.forename: Kelly
authors.initials: K
authors.lastname: Theriault
authors.forename: Erin
authors.initials: E
authors.lastname: Code
authors.forename: Cynthia
authors.initials: C
authors.lastname: Birr
authors.forename: Nick
authors.initials: N
authors.lastname: Keen
authors.forename: Huawei
authors.initials: H
authors.lastname: Chen
mesh_terms: D020816:Amino Acid Motifs; D055162:Biocatalysis; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008745:Methylation; D008958:Models, Molecular; D010455:Peptides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
chemical_list: D010455:Peptides; D016159:Tumor Suppressor Protein p53; D011495:Histone-Lysine N-Methyltransferase; C512569:SMYD2 protein, human; D015032:Zinc