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1.
Biochemistry ; 59(38): 3582-3593, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32881481

RESUMO

Substrate-induced conformational changes present in alkanesulfonate monooxygenase (SsuD) are crucial to catalysis and lead to distinct interactions between a dynamic loop region and the active site. Accelerated molecular dynamics (aMD) simulations have been carried out to examine this potential correlation by studying wild-type SsuD and variant enzymes bound with different combinations of reduced flavin (FMNH2), C4a-peroxyflavin intermediate (FMNOO-), and octanesulfonate (OCS). Three distinct mobile loop conformations were identified: "open", "closed", and "semiclosed". The substrate-free SsuD system possessed a wide opening capable of providing full access for substrates to enter the active site. Upon binding FMNH2, SsuD adopts a closed conformation that would prevent unproductive oxidation reactions in the absence of OCS. Two salt bridges, Asp111-Arg263 and Glu205-Arg271, were identified as particularly important in maintaining the closed conformation. Experimental substitution of Arg271 to Ala did not alter the catalytic activity, but the variant in the presence of reduced flavin was more susceptible to proteolytic digestion compared to wild-type. With both FMNH2 and OCS bound in SsuD, a second conformation was formed dependent upon a favorable π-π interaction between His124 and Phe261. Accordingly, there was no observed activity with the F261W SsuD variant in steady-state kinetic assays. This semiclosed conformation may be more appropriate for accepting O2 into the binding pocket and/or may properly orient the active site for the ensuing oxygenolytic cleavage. Finally, simulations of SsuD simultaneously bound with FMNOO- and OCS found an open mobile loop region that suggests alternative flavin intermediates may participate in the reaction mechanism.


Assuntos
Proteínas de Escherichia coli/química , Oxigenases de Função Mista/química , Ácidos Alcanossulfônicos/química , Ácidos Alcanossulfônicos/metabolismo , Domínio Catalítico , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Flavinas/química , Flavinas/metabolismo , Cinética , Oxigenases de Função Mista/metabolismo , Modelos Químicos , Simulação de Dinâmica Molecular , Oxirredução , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
2.
Biochemistry ; 57(8): 1349-1359, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29378138

RESUMO

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 SN2 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.


Assuntos
Proteína-Arginina N-Metiltransferases/metabolismo , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Domínio Catalítico , Metilação , Simulação de Dinâmica Molecular , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Mutação Puntual , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , Ratos , Alinhamento de Sequência , Especificidade por Substrato , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/microbiologia
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