Метаболизм D-арабинозы: характеристика бифункциональной арабинокиназы/пирофосфорилазы Leishmania major

ВЫВОДЫ.

1. Ген ImjFl6.0480 паразитического простейшего L. major кодирует бифункциональную арабинокиназу/пирофосфорилазу, способную активировать D-Ara до GDP-D-Ara через промежуточный Ara-1-P в присутствии одновременно АТР и GTP. Продукт другого гена, lmjF16.0440, лишен пирофосфорилазной активности, но способен синтезировать Ara-1-P.

2. Ферменты, кодируемые генами ImjF16.0480 и ImjFl6.0440, названные AFKP80 и FKP40, кроме D-Ara могут использовать в качестве субстрата L-Fuc, а именно, синтезировать GDP-L-Fuc в присутствии одновременно АТР и GTP.

3. Киназный домен обоих ферментов L. major расположен на С-конце, а пирофосфорилазный домен формируется при участии последовательностей, расположенных как на N-, так и на С-конце.

4. Белок AFKP80 является главным ферментом L. major, синтезирующим GDP-D-Ara.

5. GDP-D-Ara транспортируется из цитоплазмы в люмен аппарата Гольджи белком-переносчиком LPG2.

6. Синтез свободной D-Ara в цитоплазме запускает и регулирует каскад биосинтетических реакций, ведущих к арабинозилированию боковых цепей липофосфогликана Leishmania.

БЛАГОДАРНОСТИ.

Автор выражает свою признательность X. Гуо (Dr. Hongjie Guo, University of Washington, USA) за помощь в идентификации и клонировании генов Leishmania и получении гомозиготных нокаутных линий L. major fkp40(~) и L. major jkp80(-) JI. Комсток (Dr. Laurie Comstock, Harvard Medical School, USA) за предоставление плазмид, содержащих клонированные гены Bacteroides fragilis и Arabidopsis thalianaТ. Ноттон (Timothy Notton, University of Washington, USA) за помощь в проведении сайт-направленного мутагенеза гена lmjF16.0440 Т. Баррон (Tamara Barron, University of Kentucky, USA), С. Турко (Prof. Salvatore Turco, University of Kentucky, USA) и С. Беверли (Prof. Stephen Beverley, University of Washington, USA) за помощь в обсуждении результатов.

ЗАКЛЮЧЕНИЕ

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Проведенные исследования позволили охарактеризовать биосинтез D-арабинозы у паразитического простейшего Leishmania major на этапе синтеза GDP-D-Ara — донора для реакций гликозилирования. В геноме L. major были обнаружены два гена, отличающиеся по трем а.к. остаткам, один из которых {ImjF 16.0480), кодирует бифункциональную арабинокиназу-пирофосфорилазу, способную активировать D-Ara до GDP-D-Ara через промежуточный Ara-1-P в присутствии АТР и GTP. Продукт второго гена (lmjF 16.0440), не обладал пирофосфорилазной активностью и синтезировал только Ara-1-P. При этом оба фермента, AFKP80 (Arabino/FucoKinase/Pyrophosphorylase) и FKP40 (FucoKinase-Pyrophosphorylase) соответственно, обладали бифункциональной активностью со структурным гомологом D-арабинозы, L-фукозой, т. е. синтезировали GDP-L-Fuc из L-Fuc в присутствии АТР и GTP. Методом сайт-направленного мутагенеза было установлено, что киназный домен ферментов L. major расположен на С-конце, а пирофосфорилазный домен разобщен на полипетидной цепи и образуется при участии последовательностей, расположенных как на N-, так и на С-конце. Измерение кинетических параметров позволило предположить, что оба активных центра бифункциональных ферментов связаны между собой и действуют взаимозависимо, обеспечивая максимальную каталитическую активность белков. Установлено, что AFKP80 является главным ферментом, синтезирующим GDP-D-Ara в цитоплазме L. major. Нокаутирование соответствующего гена (ImjF 16.0480) приводило к прекращению арабинозилирования боковых цепей липофосфогликана клеточной поверхности Leishmania. Цитоплазматический GDP-D-Ara должен попасть в аппарат Гольджи, где происходят реакции гликозилирования. В данной работе показано, что у L. major транспорт GDP-D-Ara в аппарат Гольджи осуществляется белком переносчиком LPG2.

Таким образом, проведенное в рамках диссертационной работы исследование расширяет знания о биосинтезе редко встречающихся гликоконъюгатов — производных D-арабинопиранозы, а также, о структуре и особенностях функционирования бифункциональных киназ-пирофосфорилаз, что может быть использовано при дальнейшем изучении этого семейства ферментов.

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