[R35] Molecular mechanisms of alkane hydroxylase (AlkB) reactivity and selectivity
Ente: National Institute of General Medical Sciences
Scadenza: 2030-06-30
Importo max: 346.634 EUR
Paese: US
Descrizione
Abstract
The class-III diiron proteins, a group of integral membrane proteins unified by a histidine-rich
active site, catalyze a wide range of reactions including hydroxylations required for the production
of sphingolipids (an essential component of the myelin sheath), desaturating fatty acids that
regulate metabolism and cancer progress, and hydroxylating straight chain alkanes, enabling
them to be biodegraded in oil-impacted environments. The diverse chemistry in this enzyme
family is controlled, at least in part, by substrate channels well matched to the structure of the
different substrates. The structure of the active site, only recently determined, is puzzling because
no covalent bridge linking the two redox active iron ions is apparent. Electrons are required to
activate these enzymes. Some class-III diiron proteins have their electron-transfer partners
covalently bound while others do not. The functional significance of these different modalities is
not known. This program will target structure/function relationships in alkane monooxygenases
(AlkB), the most biochemically tractable member of the class-III diiron protein family, to
understand reaction mechanisms and the factors that control reaction scope. We will combine
mechanistic work on AlkB variants with spectroscopic characterization of the protein using a
variety of techniques that can shed light on the three dimensional and electronic structure of the
active site. We will mine our large library of functional AlkB enzymes (currently we can express
more than 60 different AlkBs from different bacteria) to search for patterns in reactivity. We will
utilize information obtained from the cryo-EM structure of AlkB we published in 2023, as well as
deep mutational scanning, to develop a large library of variant to probe the structural factors that
control reactivity. We will determine the mechanism of the recently discovered capability of AlkB
to catalyze the defluorination of fluorinated alkanes. We will attempt to determine the structure of
the new family of AlkBs whose existence we recently reported—a fusion protein containing two
electron transfer partners not previously seen linked to AlkB—and characterize its reactivity. We
will also explore whether archaea express AlkBs capable of participating in the global carbon
cycle. We expect to generate new insights into mechanisms of selective C-H and C-F bond
activation. By leveraging our unique expertise with this key member of the class-III diiron proteins,
we expect to contribute to a fundamental understanding of how these metalloenzymes work, with
implications for both human and environmental health.
Istituzione: BARNARD COLLEGE
PI: Rachel Narehood Austin
Progetto: 5R35GM158408-02
Settori: National Institute of General Medical Sciences
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