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Mutational and Functional Analyses of Substrate Binding and Catalysis of the Listeria monocytogenes EutT ATP:Co(I)rrinoid Adenosyltransferase.

Authors
  • Costa, Flavia G1
  • Greenhalgh, Elizabeth D2
  • Brunold, Thomas C2
  • Escalante-Semerena, Jorge C1
  • 1 Department of Microbiology, University of Georgia, Athens, Georgia 30602, United States. , (Georgia)
  • 2 Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United States. , (United States)
Type
Published Article
Journal
Biochemistry
Publisher
American Chemical Society
Publication Date
Mar 17, 2020
Volume
59
Issue
10
Pages
1124–1136
Identifiers
DOI: 10.1021/acs.biochem.0c00078
PMID: 32125848
Source
Medline
Language
English
License
Unknown

Abstract

ATP: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.

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