Phe71 in Type III Trypanosomal Protein Arginine Methyltransferase 7 (TbPRMT7) Restricts the Enzyme to Monomethylation

被引:21
作者
Caceres, Tamar B. [1 ]
Thakur, Abhishek [2 ]
Price, Owen M. [1 ]
Ippolito, Nicole [2 ]
Li, Jun [3 ,4 ]
Qu, Jun [3 ,4 ]
Acevedo, Orlando [2 ]
Hevel, Joan M. [1 ]
机构
[1] Utah State Univ, Dept Chem & Biochem, 0300 Old Main Hill, Logan, UT 84322 USA
[2] Univ Miami, Dept Chem, Coral Gables, FL 33146 USA
[3] Univ Buffalo State Univ New York, Dept Pharmaceut Sci, Kapoor 318,North Campus, Buffalo, NY 14260 USA
[4] New York State Ctr Excellence Bioinformat & Life, 701 Ellicott St, Buffalo, NY 14203 USA
基金
美国国家科学基金会;
关键词
ACCELERATED MOLECULAR-DYNAMICS; PRMT1 PRODUCT SPECIFICITY; ASYMMETRIC DIMETHYLARGININE; SYMMETRIC DIMETHYLARGININE; ACTIVE-SITE; METHYLATION; TRANSCRIPTION; AUTOMETHYLATION; DEPOSITION; RESIDUES;
D O I
10.1021/acs.biochem.7b01265
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Protein arginine methyltransferase 7 (PRMT7) is unique within the PRMT family as it is the only isoform known to exclusively make monomethylarginine (MMA). Given its role in epigenetics, the mechanistic basis for the strict monomethylation activity is under investigation. It is thought that PRMT7 enzymes are unable to add a second methyl group because of steric hindrance in the active site that restricts them to monomethylation. To test this, we probed the active site of trypanosomal PRMT7 (TbPRMT7) using accelerated molecular dynamics, site-directed mutagenesis, kinetic, binding, and product analyses. Both the dynamics simulations and experimental results show that the mutation of Phe71 to Ile converts the enzyme from a type III methyltransferase into a mixed type I/II, that is, an enzyme that can now perform dimethylation. In contrast, the serine and alanine mutants of Phe71 preserve the type III behavior of the native enzyme. These results are inconsistent with a sterics-only model to explain product specificity. Instead, molecular dynamics simulations of these variants bound to peptides show hydrogen bonding between would-be substrates and Glu172 of TbPRMT7. Only in the case of the Phe71 to Ile mutation is this interaction between MMA and the enzyme maintained, and the geometry for optimal S-N(2) methyl transfer is obtained. The results of these studies highlight the benefit of combined computational and experimental methods in providing a better understanding for how product specificity is dictated by PRMTs.
引用
收藏
页码:1349 / 1359
页数:11
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