bi0c00078_si_001.pdf (3.63 MB)
Mutational and Functional Analyses of Substrate Binding and Catalysis of the Listeria monocytogenes EutT ATP:Co(I)rrinoid Adenosyltransferase
journal contribution
posted on 2020-03-09, 14:33 authored by Flavia
G. Costa, Elizabeth D. Greenhalgh, Thomas C. Brunold, Jorge C. Escalante-SemerenaATP:Co(I)rrinoid
adenosyltransferases (ACATs) catalyze the transfer
of the adenosyl moiety from co-substrate ATP to a corrinoid substrate.
ACATs are grouped into three families, namely, CobA, PduO, and EutT.
The EutT family of enzymes is further divided into two classes, depending
on whether they require a divalent metal ion for activity (class I
and class II). To date, a structure has not been elucidated for either
class of the EutT family of ACATs. In this work, results of bioinformatics
analyses revealed several conserved residues between the C-terminus
of EutT homologues and the structurally characterized Lactobacillus
reuteri PduO (LrPduO) homologue. In LrPduO, these residues are associated with ATP binding and
formation of an intersubunit salt bridge. These residues were substituted,
and in vivo and in vitro data support
the conclusion that the equivalent residues in the metal-free (i.e.,
class II) Listeria monocytogenes EutT (LmEutT) enzyme affect ATP binding. Results of in vivo and in vitro analyses of LmEutT
variants with substitutions at phenylalanine and tryptophan residues
revealed that replacement of the phenylalanine residue at position
72 affected access to the substrate-binding site and replacement of
a tryptophan residue at position 238 affected binding of the Cbl substrate
to the active site. Unlike the PduO family of ACATs, a single phenylalanine
residue is not responsible for displacement of the α-ligand.
Together, these data suggest that while EutT enzymes share a conserved
ATP-binding motif and an intersubunit salt bridge with PduO family
ACATs, class II EutT family ACATs utilize an unidentified mechanism
for Cbl lower-ligand displacement and reduction that is different
from that of PduO and CobA family ACATs.