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Purification and Characterization of a Novel Selenocysteine Lyase from Enterococcus faecalis

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Date Issued:
2014
Abstract/Description:
A previous study identified Enterococcus faecalis as one of two bacteria known to have the selD gene and other selenium related genes without having the genes necessary to make selenocysteine or selenouridine. EF2570, a gene in the cluster, was later shown to be upregulated during biofilm formation and also responsible for a selenite- and molybdate-dependent increase in biofilm formation in vitro. The protein encoded was identified as a selenium dependent molybdenum hydroxylase (SDMH), enzymes that contain a labile selenium atom required for activity. While the process of inserting selenocysteine into a protein is well known, the process by which a SDMH acquires a labile selenium atom has not yet been described. To begin unraveling this pathway, the nifS-like EF2568 from the gene cluster will be characterized. Some NifS-like proteins have been shown to have selenocysteine lyase activity, providing a source of selenium for selenophosphate synthetase, the selD gene product. Study of EF2568 has shown that it specifically reacts with L-selenocysteine to form selenide and alanine with L-cysteine inhibiting the reaction. Guided by homology to the well-characterized human and E. coli NifS-like proteins, mutants of the active site and substrate discerning residues were also characterized for activity with L-selenocysteine and L-cysteine. While mutation of the residue at position 112 thought to be responsible for substrate specificity did not affect reactivity of the enzyme with L-cysteine, it did affect reactivity with L-selenocysteine. Studying the characteristics of this novel group II selenocysteine lyase will provide a foundation for studying the remaining pathway.
Title: Purification and Characterization of a Novel Selenocysteine Lyase from Enterococcus faecalis.
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Name(s): Nelson, Samantha, Author
Self, William, Committee Chair
Moore, Sean, Committee Member
Rohde, Kyle, Committee Member
University of Central Florida, Degree Grantor
Type of Resource: text
Date Issued: 2014
Publisher: University of Central Florida
Language(s): English
Abstract/Description: A previous study identified Enterococcus faecalis as one of two bacteria known to have the selD gene and other selenium related genes without having the genes necessary to make selenocysteine or selenouridine. EF2570, a gene in the cluster, was later shown to be upregulated during biofilm formation and also responsible for a selenite- and molybdate-dependent increase in biofilm formation in vitro. The protein encoded was identified as a selenium dependent molybdenum hydroxylase (SDMH), enzymes that contain a labile selenium atom required for activity. While the process of inserting selenocysteine into a protein is well known, the process by which a SDMH acquires a labile selenium atom has not yet been described. To begin unraveling this pathway, the nifS-like EF2568 from the gene cluster will be characterized. Some NifS-like proteins have been shown to have selenocysteine lyase activity, providing a source of selenium for selenophosphate synthetase, the selD gene product. Study of EF2568 has shown that it specifically reacts with L-selenocysteine to form selenide and alanine with L-cysteine inhibiting the reaction. Guided by homology to the well-characterized human and E. coli NifS-like proteins, mutants of the active site and substrate discerning residues were also characterized for activity with L-selenocysteine and L-cysteine. While mutation of the residue at position 112 thought to be responsible for substrate specificity did not affect reactivity of the enzyme with L-cysteine, it did affect reactivity with L-selenocysteine. Studying the characteristics of this novel group II selenocysteine lyase will provide a foundation for studying the remaining pathway.
Identifier: CFE0005388 (IID), ucf:50455 (fedora)
Note(s): 2014-08-01
M.S.
Medicine, Molecular Biology and Micro
Masters
This record was generated from author submitted information.
Subject(s): Enterococcus faecalis -- selenocysteine lyase -- cysteine desulfurase -- SDMH -- Moco
Persistent Link to This Record: http://purl.flvc.org/ucf/fd/CFE0005388
Restrictions on Access: campus 2015-08-15
Host Institution: UCF

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