Cytochrome P450 CYP153A
M.aq
from Marinobacter aquaeolei serves as a model enzyme for the
terminal (ω-) hydroxylation of medium- to long-chain fatty acids.
We have engineered this enzyme using different mutagenesis approaches
based on structure-sequence-alignments within the 3DM database and
crystal structures of CYP153A
M.aq
and
a homologue CYP153A
P.sp
. Applying these
focused mutagenesis strategies and site-directed saturation mutagenesis,
we created a variant that ω-hydroxylates octanoic acid. The
M.aqRLT variant exhibited 151-fold improved catalytic efficiency and
showed strongly improved substrate binding (25-fold reduced K
m compared to the wild type). We then used molecular
dynamics simulations to gain deeper insights into the dynamics of
the protein. We found the tunnel modifications and the two loop regions
showing greatly reduced flexibility in the engineered variant were
the main features responsible for stabilizing the enzyme–substrate
complex and enhancing the catalytic efficiency. Additionally, we showed
that a previously known fatty acid anchor (Q129R) interacts significantly
with the ligand to hold it in the reactive position, thereby boosting
the activity of the variant M.aqRLT toward octanoic acid. The study
demonstrates the significant effects of both substrate stabilization
and the impact of enzyme flexibility on catalytic efficiency. These
results could guide the future engineering of enzymes with deeply
buried active sites to increase or even establish activities toward
yet unknown types of substrates.
The catalytic space of the P450 monooxygenase CYP153AM.aq was opened from a terminal (ω‐) fatty acid hydroxylase to a catalyst capable of performing ω‐hydroxylation of dodecylamine, which is a potent inhibitor for the wild‐type enzyme. A simple screening method named Rapid‐flow Analysis of Product Peaks (RAPP) was established and applied to measure saturation libraries directly from a 96‐deepwell plate in 36 seconds per sample. The obtained variants are less inhibited by the amine, although concurrently show less affinity towards the acid. Molecular modelling and molecular dynamics simulations showed significant effects of the mutations on the substrate tunnel architectures.
In enzymes, the active site is the location where substrates are chemically converted. If this site is deeply buried within the protein, substrates must pass not only through the body of the protein via a tunnel, but also flexible, site-decorating loops to access the active site. These elements can act as filters that influence on both substrate specificity and activity. Identifying and understanding how they exert such control has been of growing interest over the past several years.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.