Sulfur-Element containing metabolic pathways in human health and crosstalk with the microbiome.

In humans, methionine derived from dietary proteins is necessary for cellular homeostasis and regeneration of sulfur containing pathways, which produce inorganic sulfur species (ISS) along with essential organic sulfur compounds (OSC). In recent years, inorganic sulfur species have gained attention as key players in the crosstalk of human health and the gut microbiome. Endogenously, ISS includes hydrogen sulfide (H2S), sulfite (SO32-), thiosulfate (S2O32-), and sulfate (SO42-), which are produced by enzymes in the transsulfuration and sulfur oxidation pathways. Additionally, sulfate-reducing bacteria (SRB) in the gut lumen are notable H2S producers which can contribute to the ISS pools of the human host. In this review, we will focus on the systemic effects of sulfur in biological pathways, describe the contrasting mechanisms of sulfurylation versus phosphorylation on the hydroxyl of serine/threonine and tyrosine residues of proteins in post-translational modifications, and the role of the gut microbiome in human sulfur metabolism.

Gamma cleavage is a rate-determining step in the gamma-elimination reaction of L-methionine analogues catalyzed by methionine-gamma-lyase.

Methionine-γ-lyase (MGL) is a pyridoxal-5'-phosphate dependent enzyme found in bacteria and protozoa that catalyzes a variety of reactions, including the γ-elimination of L-methionine (L-Met). Here we report experimental kinetic data and density functional theory (DFT) computational data for the γ-elimination reaction of L-Met and several other substrate analogues by a recombinant MGL from P. gingivalis (MGL_Pg). UV-Visible spectrophotometry experiments revealed a heavily populated species with maximum absorbance at 478 nm during steady-state catalysis of L-Met, L-ethionine, L-methionine sulfone and L-homoserine, which we assign to a late crotonate intermediate formed after the γ-cleavage step in the reaction and thus common to all substrates. A more red-shifted (498 nm) species was observed during the reaction of L-homoserine lactone, which we assign to an early quinonoid intermediate with the aid of time-dependent self-consistent field calculations. Significant differences in both binding and the rate of turnover were observed for the substrates. MGL_Pg's highest catalytic efficiency was recorded for L-vinylglycine (kcat/Km = 6455 s-1 M-1), exceeding that of L-Met (kcat/Km = 4211 s-1 M-1), while L-Met sulfone displayed the largest turnover number (kcat = 1638 min-1). A direct correlation between experimental kcat values and DFT-calculated γ-cleavage Gibbs activation energies was identified for the various substrates. In light of these data, we propose that the γ-cleavage step in the catalytic reaction pathway is rate-limiting. This conclusion has direct implications for the rational design of substrates or inhibitors aimed at regulating MGL activity.

A continuous spectrophotometric assay and nonlinear kinetic analysis of methionine γ-lyase catalysis.

In this article, we present a new, easy-to-implement assay for methionine γ-lyase (MGL)-catalyzed γ-elimination reactions of l-methionine and its analogues that produce α-ketobutyrate (α-KB) as product. The assay employs ultraviolet-visible (UV-Vis) spectrophotometry to continuously monitor the rate of formation of α-KB by its absorbance at 315 nm. We also employ a nonlinear data analysis method that obviates the need for an "initial slope" determination, which can introduce errors when the progress curves are nonlinear. The spectrophotometric assay is validated through product analysis by (1)H NMR (nuclear magnetic resonance), which showed that under the conditions of study l-methionine (l-met) and l-methionine sulfone (l-met sulfone) substrates were converted to α-KB product with greater than 99% yield. Using this assay method, we determined for the first time the Michaelis-Menten parameters for a recombinant form of MGL from Porphyromonas gingivalis, obtaining respective kcat and Km values of 328 ± 8 min(-1) and 1.2 ± 0.1 mM for l-met γ-elimination and 2048 ± 59 min(-1) and 38 ± 2 mM for l-met sulfone γ-elimination reactions. We envisage that this assay method will be useful for determining the activity of MGL γ-elimination reactions that produce α-KB as the end product.