Characterization of C-S lyase fromLactobacillus delbrueckiisubsp.bulgaricusATCC BAA-365 and its potential role in food flavour applications

Abstract

Volatile thiols have substantial impact on the aroma of many beverages and foods. Thus, the control of their formation, which has been linked to C-S lyase enzymatic activities, is of great significance in industrial applications involving food flavours. Herein, we have carried out a spectroscopic and functional characterization of a putative pyridoxal 5'-phosphate (PLP)-dependent C-S lyase from the lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 (LDB C-S lyase). Recombinant LDB C-S lyase exists as a tetramer in solution and shows spectral properties of enzymes containing PLP as cofactor. The enzyme has a broad substrate specificity toward sulphur-containing amino acids with aminoethyl-L-cysteine and L-cystine being the most effective substrates over L-cysteine and L-cystathionine. Notably, the protein also reveals cysteine-S-conjugate β-lyase activity in vitro, and is able to cleave a cysteinylated substrate precursor into the corresponding flavour-contributing thiol, with a catalytic efficiency higher than L-cystathionine. Contrary to similar enzymes of other lactic acid bacteria however, LDB C-S lyase is not capable of α,γ-elimination activity towards L-methionine to produce methanethiol, which is a significant compound in flavour development. Based on our results, future developments can be expected regarding the flavour-forming potential of Lactobacillus C-S lyase and its use in enhancing food flavours.

Snapshots of C-S Cleavage in Egt2 Reveals Substrate Specificity and Reaction Mechanism

“…In the Egt2-catalyzed C-S lyase reaction of hercynylcysteine sulfoxide 4 in the presence of DTT, ergothioneine, pyruvate and ammonia were produced in a ratio of ~ 1:1:1. Taking advantage of pyruvate production and reaction stoichiometry, the kinetic properties of wild-type Egt2 were subsequently measured using a colorimetric assay by coupling wild-type Egt2 catalysis with the lactate dehydrogenase reaction (Song et al, 2015, Allegrini et al, 2017, Pioselli et al, 2004). When sulfoxide 4 was used as a substrate and in the presence of DTT at pH 8.0, the kinetic parameters of wild-type Egt2 were determined to be k cat = 8.7 ± 0.1 s −1 and K m = 155 ± 10 μM (Fig.…”

Cystathionine β-synthase is involved in cysteine biosynthesis and H2S generation in Toxoplasma gondii

“…High molecular weight gel filtration calibration kit (GE Healthcare) was used to construct a calibration curve, following protocols in Refs. 50,51 .…”

Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii

“…Enzyme activity was determined using a Jasco-V560 UV-Vis spectrophotometer (Jasco Europe, Cremella (LC), Italy) via two spectrophotometric assays: the reaction of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) with the free thiol of the product ( ε 412 = 13600 M −1 ·cm −1 ) and by monitoring the pyruvate formation with the coupling enzyme NADH-dependent lactate dehydrogenase (LDH) ( ε 340 = 6200 M −1 ·cm −1 ), as described [ 34 , 35 ]. All enzymatic assays were carried out at 37 °C in 50 mM MOPS, bicine, proline (MBP) buffer pH 9 in the presence of 20 μM PLP.…”

“…Apo-proteins were obtained following the protocol in [ 35 , 37 ]. The apo-proteins showed no absorption peaks in the region 300–500 nm and no residual activity.…”