vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/vjgb-25-111vavilov-4889Research ArticleСТРУКТУРНАЯ КОМПЬЮТЕРНАЯ БИОЛОГИЯSTRUCTURAL COMPUTATIONAL BIOLOGYМолекулярно-динамический анализ функциональной роли аминокислотных остатков V99, F124 и S125 ДНК-диоксигеназы человека ABH2Molecular dynamic analysis of the functional role of amino acid residues V99, F124 and S125 of human DNA dioxygenase ABH2https://orcid.org/0009-0005-6712-8206ЧжаоМ.ZhaoM.

Новосибирск

Novosibirsk

https://orcid.org/0000-0002-2539-2894ТюгашевТ. Е.TyugashevT. E.

Новосибирск

Novosibirsk

https://orcid.org/0000-0002-4419-3954ДавлетгильдееваА. Т.DavletgildeevaA. T.

Новосибирск

Novosibirsk

https://orcid.org/0000-0002-4016-198XКузнецовН. А.KuznetsovN. A.

Новосибирск

Novosibirsk

kuznetsov@1bio.ruНовосибирский национальный исследовательский государственный университетРоссияNovosibirsk State UniversityRussian FederationИнститут химической биологии и фундаментальной медицины Сибирского отделения Российской академии наукРоссияInstitute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of SciencesRussian FederationНовосибирский национальный исследовательский государственный университет; Институт химической биологии и фундаментальной медицины Сибирского отделения Российской академии наукРоссияNovosibirsk State University; Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of SciencesRussian Federation20251212202529710621072Copyright © Чжао М., Тюгашев Т.Е., Давлетгильдеева А.Т., Кузнецов Н.А., 20252025Чжао М., Тюгашев Т.Е., Давлетгильдеева А.Т., Кузнецов Н.А.Zhao M., Tyugashev T.E., Davletgildeeva A.T., Kuznetsov N.A.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/4889

   ДНК-диоксигеназа человека ABH2 относится к семейству AlkB-подобных негемовых диоксигеназ, которые действуют на широкий спектр субстратов и обладают сложным каталитическим механизмом с участием α-кетоглутарата и иона Fe(II) в качестве кофактора. Представители семейства AlkB катализируют прямое окисление алкильных заместителей в азотистых основаниях ДНК и РНК, обеспечивая защиту от мутагенного воздействия эндогенных и экзогенных алкилирующих агентов, а также участвуя в регуляции уровня метилирования некоторых РНК. Фермент ABH2, локализованный преимущественно в ядре клетки, проявляет специфичность к двуцепочечным ДНК-субстратам и, в отличие от большинства других AlkB-подобных ферментов человека, обладает довольно широким спектром субстратной специфичности, окисляя алкильные группы таких модифицированных азотистых оснований, как, например, N 1-метиладенозин, N 3-метилцитидин, 1,N 6-этеноаденозин и 3,N 4-этеноцитидин.

   В данной работе с целью анализа механизма, обеспечивающего субстратную специфичность фермента, и выяснения функциональной роли аминокислотных остатков в составе активного центра нами выполнено молекулярно-динамическое моделирование комплексов фермента ABH2 дикого типа и его мутантных форм, содержащих аминокислотные замены V99A, F124A или S125A, с двумя типами ДНК-субстратов, несущих метилированные основания N 1-метиладенин или N 3-метилцитозин.

   Установлено, что замена V99A приводит к увеличению подвижности белковых петель L1 и L2, участвующих в связывании ДНК-субстрата, и изменяет распределение π-π-контактов боковой цепи остатка F102 с азотистыми основаниями, расположенными рядом с поврежденным нуклеотидом. Замена F124A приводит к потере π-π-стэкинга с поврежденным основанием, что, в свою очередь, дестабилизирует архитектуру активного центра, вызывает нарушение взаимодействия с ионом железа и препятствует оптимальному каталитическому позиционированию α-кетоглутарата в активном центре. Замена S125A приводит к потере прямого взаимодействия петли L2 с 5’-фосфатной группой поврежденного нуклеотида, ослабляя связывание фермента с ДНК-субстратом. Таким образом, полученные данные позволили установить функциональную роль трех аминокислотных остатков активного центра и расширить понимание структурно-функциональных связей в процессах узнавания поврежденного нуклеотида и формирования каталитического комплекса ферментом ABH2 человека.

   The ABH2 enzyme belongs to the AlkB-like family of Fe(II)/α-ketoglutarate-dependent dioxygenases. Various non-heme dioxygenases act on a wide range of substrates and have a complex catalytic mechanism involving α-ketoglutarate and an Fe(II) ion as a cofactor. Representatives of the AlkB family catalyze the direct oxidation of alkyl substituents in the nitrogenous bases of DNA and RNA, providing protection against the mutagenic effects of endogenous and exogenous alkylating agents, and also participate in the regulation of the methylation level of some RNAs. DNA dioxygenase ABH2, localized predominantly in the cell nucleus, is specific for double-stranded DNA substrates and, unlike most other human AlkB-like enzymes, has a fairly broad spectrum of substrate specificity, oxidizing alkyl groups of such modified nitrogenous bases as, for example, N 1-methyladenosine, N 3-methylcytidine, 1,N 6-ethenoadenosine and 3,N 4-ethenocytidine.

   To analyze the mechanism underlying the enzyme’s substrate specificity and to clarify the functional role of key active-site amino acid residues, we performed molecular dynamics simulations of complexes of the wild-type ABH2 enzyme and its mutant forms containing amino acid substitutions V99A, F124A and S125A with two types of DNA substrates carrying methylated bases N 1-methyladenine and N 3-methylcytosine, respectively.

   It was found that the V99A substitution leads to an increase in the mobility of protein loops L1 and L2 involved in binding the DNA substrate and changes the distribution of π-π contacts between the side chain of residue F102 and nitrogenous bases located near the damaged nucleotide. The F124A substitution leads to the loss of π-π stacking with the damaged base, which in turn destabilizes the architecture of the active site, disrupts the interaction with the iron ion and prevents optimal catalytic positioning of α-ketoglutarate in the active site. The S125A substitution leads to the loss of direct interaction of the L2 loop with the 5’-phosphate group of the damaged nucleotide, weakening the binding of the enzyme to the DNA substrate. Thus, the obtained data revealed the functional role of three amino acid residues of the active site and contributed to the understanding of the structural-functional relationships in the recognition of a damaged nucleotide and the formation of a catalytic complex by the human ABH2 enzyme.

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The authors declare that there are no conflicts of interest present.