An NAD+-dependent sirtuin depropionylase and deacetylase (Sir2La) from the probiotic bacterium Lactobacillus acidophilus NCFM

Sita V. Olesen, Nima Rajabi, Birte Svensson, Christian A. Olsen, Andreas S. Madsen*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Sirtuins, a group of NAD+-dependent deacylases-have emerged as key in the connection between NAD+ metabolism and aging. This class of enzymes hydrolyze a range of ε- N-acyllysine PTMs and determining the repertoire of catalyzed deacylation reactions is of high importance to fully elucidate the roles of a given sirtuin. Here we have identified and produced two potential sirtuins from the probiotic bacterium Lactobacillus acidophilus NCFM and screening more than 80 different substrates, covering 26 acyl groups on five peptide scaffolds, demonstrated that one of the investigated proteins-Sir2La-is a bona fide NAD+-dependent sirtuin, catalyzing hydrolysis of acetyl-, propionyl-, and butyryllysine. Further substantiating the identity of Sir2La as a sirtuin, known sirtuin inhibitors nicotinamide and suramin as well as a thioacetyllysine compound inhibit the deacylase activity in a concentration-dependent manner. Based on steady-state kinetics Sir2La showed a slight preference for propionyllysine (Kpro) over acetyllysine (Kac). For non-fluorogenic peptide substrates the preference is driven by a remarkably low KM (280 nM vs 700 nM, for Kpro and Kac, respectively) whereas kcat was similar (21 X 10-3 s-1). Moreover, while NAD+ is a prerequisite for Sir2La-mediated deacylation, Sir2La has very high KM for NAD+ compared to the expected levels of the dinucleotide in L. acidophilus. Sir2La is the first sirtuin from Lactobacillales and of the Gram-positive bacterial subclass of sirtuins to be functionally characterized. The ability to hydrolyze propionyl- and butyryllysine emphasizes the relevance of further exploring the role of other short-chain acyl moieties as PTMs.
Original languageEnglish
JournalBiochemistry
Volume57
Issue number26
Pages (from-to)3903-3915
ISSN0006-2960
DOIs
Publication statusPublished - 2018

Cite this

Olesen, Sita V. ; Rajabi, Nima ; Svensson, Birte ; Olsen, Christian A. ; Madsen, Andreas S. / An NAD+-dependent sirtuin depropionylase and deacetylase (Sir2La) from the probiotic bacterium Lactobacillus acidophilus NCFM. In: Biochemistry. 2018 ; Vol. 57, No. 26. pp. 3903-3915.
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title = "An NAD+-dependent sirtuin depropionylase and deacetylase (Sir2La) from the probiotic bacterium Lactobacillus acidophilus NCFM",
abstract = "Sirtuins, a group of NAD+-dependent deacylases-have emerged as key in the connection between NAD+ metabolism and aging. This class of enzymes hydrolyze a range of ε- N-acyllysine PTMs and determining the repertoire of catalyzed deacylation reactions is of high importance to fully elucidate the roles of a given sirtuin. Here we have identified and produced two potential sirtuins from the probiotic bacterium Lactobacillus acidophilus NCFM and screening more than 80 different substrates, covering 26 acyl groups on five peptide scaffolds, demonstrated that one of the investigated proteins-Sir2La-is a bona fide NAD+-dependent sirtuin, catalyzing hydrolysis of acetyl-, propionyl-, and butyryllysine. Further substantiating the identity of Sir2La as a sirtuin, known sirtuin inhibitors nicotinamide and suramin as well as a thioacetyllysine compound inhibit the deacylase activity in a concentration-dependent manner. Based on steady-state kinetics Sir2La showed a slight preference for propionyllysine (Kpro) over acetyllysine (Kac). For non-fluorogenic peptide substrates the preference is driven by a remarkably low KM (280 nM vs 700 nM, for Kpro and Kac, respectively) whereas kcat was similar (21 X 10-3 s-1). Moreover, while NAD+ is a prerequisite for Sir2La-mediated deacylation, Sir2La has very high KM for NAD+ compared to the expected levels of the dinucleotide in L. acidophilus. Sir2La is the first sirtuin from Lactobacillales and of the Gram-positive bacterial subclass of sirtuins to be functionally characterized. The ability to hydrolyze propionyl- and butyryllysine emphasizes the relevance of further exploring the role of other short-chain acyl moieties as PTMs.",
author = "Olesen, {Sita V.} and Nima Rajabi and Birte Svensson and Olsen, {Christian A.} and Madsen, {Andreas S.}",
year = "2018",
doi = "10.1021/acs.biochem.8b00306",
language = "English",
volume = "57",
pages = "3903--3915",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
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An NAD+-dependent sirtuin depropionylase and deacetylase (Sir2La) from the probiotic bacterium Lactobacillus acidophilus NCFM. / Olesen, Sita V.; Rajabi, Nima; Svensson, Birte; Olsen, Christian A.; Madsen, Andreas S.

In: Biochemistry, Vol. 57, No. 26, 2018, p. 3903-3915.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - An NAD+-dependent sirtuin depropionylase and deacetylase (Sir2La) from the probiotic bacterium Lactobacillus acidophilus NCFM

AU - Olesen, Sita V.

AU - Rajabi, Nima

AU - Svensson, Birte

AU - Olsen, Christian A.

AU - Madsen, Andreas S.

PY - 2018

Y1 - 2018

N2 - Sirtuins, a group of NAD+-dependent deacylases-have emerged as key in the connection between NAD+ metabolism and aging. This class of enzymes hydrolyze a range of ε- N-acyllysine PTMs and determining the repertoire of catalyzed deacylation reactions is of high importance to fully elucidate the roles of a given sirtuin. Here we have identified and produced two potential sirtuins from the probiotic bacterium Lactobacillus acidophilus NCFM and screening more than 80 different substrates, covering 26 acyl groups on five peptide scaffolds, demonstrated that one of the investigated proteins-Sir2La-is a bona fide NAD+-dependent sirtuin, catalyzing hydrolysis of acetyl-, propionyl-, and butyryllysine. Further substantiating the identity of Sir2La as a sirtuin, known sirtuin inhibitors nicotinamide and suramin as well as a thioacetyllysine compound inhibit the deacylase activity in a concentration-dependent manner. Based on steady-state kinetics Sir2La showed a slight preference for propionyllysine (Kpro) over acetyllysine (Kac). For non-fluorogenic peptide substrates the preference is driven by a remarkably low KM (280 nM vs 700 nM, for Kpro and Kac, respectively) whereas kcat was similar (21 X 10-3 s-1). Moreover, while NAD+ is a prerequisite for Sir2La-mediated deacylation, Sir2La has very high KM for NAD+ compared to the expected levels of the dinucleotide in L. acidophilus. Sir2La is the first sirtuin from Lactobacillales and of the Gram-positive bacterial subclass of sirtuins to be functionally characterized. The ability to hydrolyze propionyl- and butyryllysine emphasizes the relevance of further exploring the role of other short-chain acyl moieties as PTMs.

AB - Sirtuins, a group of NAD+-dependent deacylases-have emerged as key in the connection between NAD+ metabolism and aging. This class of enzymes hydrolyze a range of ε- N-acyllysine PTMs and determining the repertoire of catalyzed deacylation reactions is of high importance to fully elucidate the roles of a given sirtuin. Here we have identified and produced two potential sirtuins from the probiotic bacterium Lactobacillus acidophilus NCFM and screening more than 80 different substrates, covering 26 acyl groups on five peptide scaffolds, demonstrated that one of the investigated proteins-Sir2La-is a bona fide NAD+-dependent sirtuin, catalyzing hydrolysis of acetyl-, propionyl-, and butyryllysine. Further substantiating the identity of Sir2La as a sirtuin, known sirtuin inhibitors nicotinamide and suramin as well as a thioacetyllysine compound inhibit the deacylase activity in a concentration-dependent manner. Based on steady-state kinetics Sir2La showed a slight preference for propionyllysine (Kpro) over acetyllysine (Kac). For non-fluorogenic peptide substrates the preference is driven by a remarkably low KM (280 nM vs 700 nM, for Kpro and Kac, respectively) whereas kcat was similar (21 X 10-3 s-1). Moreover, while NAD+ is a prerequisite for Sir2La-mediated deacylation, Sir2La has very high KM for NAD+ compared to the expected levels of the dinucleotide in L. acidophilus. Sir2La is the first sirtuin from Lactobacillales and of the Gram-positive bacterial subclass of sirtuins to be functionally characterized. The ability to hydrolyze propionyl- and butyryllysine emphasizes the relevance of further exploring the role of other short-chain acyl moieties as PTMs.

U2 - 10.1021/acs.biochem.8b00306

DO - 10.1021/acs.biochem.8b00306

M3 - Journal article

VL - 57

SP - 3903

EP - 3915

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 26

ER -