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\begin{document}

\title{M42 aminopeptidase catalytic site: the structural and functional role of
a strictly conserved aspartate residue}



\author[1]{Raphael Dutoit}%
\author[2]{Nathalie Brandt}%
\author[3]{Tom Van Gompel}%
\author[4]{Dany Van Elder}%
\author[3]{Jeroen Van Dyck}%
\author[5]{Frank Sobott}%
\author[4]{Louis Droogmans}%
\affil[1]{Université Libre de Bruxelles}%
\affil[2]{ULB LABIRIS}%
\affil[3]{Universiteit Antwerpen}%
\affil[4]{ULB}%
\affil[5]{University of Leeds}%


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  \date{June 15, 2024}


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\begin{abstract}
The M42 aminopeptidases are a family of dinuclear aminopeptidases widely
distributed in Prokaryotes. They are potentially associated to the
proteasome, achieving complete peptide destruction. Their most peculiar
characteristic is their quaternary structure, a tetrahedron-shaped
particle made of twelve subunits. The catalytic site of M42
aminopeptidases is defined by seven conserved residues. Five of them are
involved in metal ion binding which is important to maintain both the
activity and the oligomeric state. The sixth conserved residue, a
glutamate, is the catalytic base deprotonating the water molecule during
peptide bond hydrolysis. The seventh residue is an aspartate whose
function remains poorly understood. This aspartate residue, however,
must have a critical role as it is strictly conserved in all MH clan
enzymes. It forms some kind of catalytic triad with the histidine
residue and the metal ion of the M2 binding site. We assess its role in
TmPep1050, an M42 aminopeptidase of \emph{Thermotoga maritima}, through
a mutational approach. Asp-62 was substituted with alanine, asparagine,
or glutamate residue. The three Asp-62 substitutions completely
abolished TmPep1050 activity and impeded dodecamer formation. They also
interfered with metal ion binding as only one cobalt ion is bound per
subunit instead of two. The structural data showed that the Asp62Ala
substitution has an impact on the active site folds becoming similar to
TmPep1050 dimer. We propose a structural role for Asp-62, helping to
stabilize a crucial loop in the active site and to position correctly
the catalytic base and a metal ion ligand of the M1 site.%
\end{abstract}%



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