No abstract

Sirtuins, a group of NAD-dependent deacylases, have emerged as the key connection between NAD metabolism and aging. This class of enzymes hydrolyzes 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. 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. On the basis of steady-state kinetics, Sir2La showed a slight preference for propionyllysine (Kpro) over acetyllysine (Kac). For nonfluorogenic peptide substrates, the preference is driven by a remarkably low K (280 nM vs 700 nM, for Kpro and Kac, respectively), whereas k was similar (21 × 10 s). Moreover, while NAD is a prerequisite for Sir2La-mediated deacylation, Sir2La has a very high K 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.

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, showed 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 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 over acetyllysine and butyryllysine, driven both by KM (14 µM vs 21 µM and 15 µM) and kcat (4.4·10 -3 s -1 vs 2.5·10 -3 s -1 and 1.21·10 -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.Nicotinamide adenosine dinucleotide (NAD + ) is well known for its role-together with the reduced form, NADH-as an essential redox couple in metabolism. While the dinucleotide is intact during oxidoreductase reactions, NAD + also serves as substrate in reactions where the charged nicotinamide moiety acts as leaving group rather than hydride acceptor. NAD + -consuming enzymes include deacylases (sirtuins), cyclic adenosine diphosphate synthetases (cADPRS), and poly(adenosine diphosphate ribose) polymerases/adenosine diphosphate-ribosyl transferases (PARPs/ARTs). Dinucleotide turnover resulting from the activity of these enzymes leaves cells in constant need of NAD + -supply. In mammals, NAD + is synthesized either via a de novo pathway from tryptophan, or via salvaging the three precursor vitamins nicotinic acid, nicotinamide, or nicotinamide riboside (NR). The recommended dietary allowance of vitamin B3 (niacin, a generic term for nicotinic acid and nicotinamide) is 15−20 mg/day, an amount sufficient to prevent clinical symptoms of niacin deficiency. However, in recent years significant interest has been raised around the use of NAD + -precursors as antiaging agents All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/252379 doi: bioRxiv...

Protein-protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultrahigh affinity interactions (K d <0.1 nM) occur in these systems, however, structures and energetics behind stability of ultra-high affinity protein-protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra-high affinity complex (K d of 42 pM). In this study the LD-LDI complex is investigated to unveil how robust the ultra-high affinity is to LDI sequence variation at the protein-protein interface and whether alternative sequences can retain the ultra-high binding affinity. The interface of LD-LDI was engineered using computational protein redesign aiming at identifying LDI variants predicted to retain ultra-high binding affinity. These variants present a very diverse set of mutations going beyond conservative and alanine substitutions typically used to probe interfaces. Surface plasmon resonance analysis of the LDI variants revealed that high affinity of LD-LDI requires interactions of several residues at the rim of the protein interface, unlike the classical hotspot arrangement where key residues are found at the center of the interface. Notably, substitution of interface residues in LDI, including amino acids with functional groups different from the wild-type, could occur without loss of affinity. This demonstrates that ultra-high binding affinity can be conferred without hotspot residues, thus making complexes more robust to mutational drift in evolution. The present mutational analysis also demonstrates how energetic coupling can emerge between residues at large distances at the interface.

The present study describes the growth of the very well-known probiotic bacterium Lactobacillus acidophilus NCFM on different carbohydrates. Furthermore, recombinant production of putative moonlighting proteins elongation factor G and pyruvate kinase from this bacterium is described. For further and detailed interpretation of the data presented here, please see the research article “Mucin- and carbohydrate-stimulated adhesion and subproteome changes of the probiotic bacterium Lactobacillus acidophilus NCFM” (Celebioglu et al., 2017) [1].