Latest revision as of 14:15, 17 December 2020

LuxS

LuxS is a synthase that produces DPD, which spontaneously forms the AI-2 signaling molecule.

S-ribosylhomocysteine lyase (EC 4.4.1.21) is a metalloenzyme that cleaves the thiol ester bond in RHC and produces homocysteine and DPD, a precursor of AI-2.

Introduction

Vilnius-Lithuania iGEM 2020 project FlavoFlowincludes three goals towards looking for Flavobacterium disease-related problems’ solutions. The project includes creating a rapid detection kit, based on HDA and LFA, developing an implement for treating a disease, and introducing the foundation of edible vaccines. This part was used for the second goal- treatment - of the project FlavoFlow. This part if for contribution.

Biology

S-ribosylhomocysteine lyase is involved in the synthesis of autoinducer-2 (AI-2), a signaling molecule secreted by bacteria. This phenomenon, when gene expression mechanisms are regulated corresponding to cell density, is called quorum sensing. Mostly regulated genes are involved in antibiotic resistance, biofilm formation, conjugation, swarming processes. The bacterial domain has a way to metabolite/recycle SAH by Pfs and LuxS or SAH-hydrolase^[1]. Recent studies show that S-ribosylhomocysteinase is coded by the highly conserved luxS gene and is present in the majority of bacteria^[2]. LuxS protein catalyzes the transformation of S-ribosyl homocysteine (RHC) to homocysteine (HC) and 4,5-dihydroxy-2,3-pentanedione (DPD).

AI-2 synthesis:

S-(5-deoxy-D-ribose-5-yl)-L-homocysteine → (S)-4,5-dihydroxypentane-2,3-dione + L-homocysteine

Autoinducer 2 is proposed to be a universal bacterial communication molecule 3 unlike the AI-1, which is species-specific. E. coli has a set of genes called lsr (luxs regulated) which are responsible for uptake and synthesis of AI-2.

The studies of crystal structures show that LuxS exists as a homodimer and has two identical active sites at the dimer interface by residues from both subunits. LuxS active sites contain a divalent metal ion Zn²⁺ or Fe²⁺. The metal ion is tetrahedrally coordinated by the side chains of histidines 54 and 58 in the HXXEH motif and Cys126, and a water molecule. The Fe²⁺ oxidizes the cysteine and causes the modification. This process inactivates the enzyme. The active sites contain Ser6, Phe7, His11, and Arg39 residues. This kind of ligand environment is very similar to that of peptide deformylase. LuxS enzyme like the deformylase loses activity at aerobic conditions due to oxidation^[3].

Despite that LuxS contains Fe²⁺ ion, tetrahedrally coordinated, S-ribosylhomocysteine‘s ion can be substituted by Co²⁺ without any changes in catalytic properties. Co²⁺ as an ion provides a more stable enzyme than native LuxS^[2]. Pei and his colleagues proposed a catalytic mechanism of LuxS. It shows that S-ribosyl homocysteine acts as a Lewis acid, expediting the aldose-ketose isomerization steps^[3].

Results

Before starting to experiment with autoinducer-2 reactions and AI-2 induced constructs,we looked at the iGEM part registry. Enzymatic characterization of S-ribosylhomocysteine lyase (luxS) was not to be found there. Therefore, our team decided to find Km and Vmax of these proteins. Traditionally, a method that determines reaction velocity is calculated using different substrate concentrations. This way, a lot of substrate would be used. Due to a shortage of S-Adenosyl-l-homocysteine (SAH) substrate, we wanted to find other methods to achieve our goal. It was found that there is a possibility to determine K_m and V_max values with single substrate concentration only. Traditionally, K_m and V_max are calculated by the equation (1) but in 1997 Schnell and Mendoza identified that the solution to the first differential eqn. (1) is in the form of equation (2)¹.

   v = - Δ[S]/Δt = Vmax[S]/Km + [S]...(Equation 1) 

Here [S] – substrate concentration, V_max is the limiting rate under saturation conditions, K_m is Michaelis constant, v is velocity of the reaction and t – reaction time.

Solution for x cannot be found from equation (2) algebraically, so various linearization procedures of (1) equation were employed for the determination of kinetic parameters.

   x = yey 	...(Equation 2) 

Solution of equation (2) can be expressed in terms of Lambert W function:

   y = Wo(x)  ...(Equation 3) 

Lambert W function is a multivalued, transcendental equation, which has applications in many scientific fields, including biochemistry. K_m and V_max parameters can be found by plotting product concentration versus reaction time and fitting equation (4) to find a global solution. Change of product - 2-nitro-5-thiobenzoate (TNB⁻) absorption was observed spectrophotometrically at 412 nm wavelength. Absorbance was recalculated to concentration according to Lambert-Beer’s equation (5).

   [P] = [S]0 - Km Wo([S]0Kmexp[S]0 - Vmaxt)/Km ...(Equation 4) 

Here [S]_o– initial substrate concentration, t – reaction time.

   A = ε412cl ...(Equation 5) 

Here ε₄₁₂ – extinction coefficient at 412 nm wavelength (14150 M^-1cm^-1), l – optical path length (1 cm), c – concentration of TNB^-.

Procedure and results

In order to calculate K_m and V_max of LuxS, we proceeded to experiment with sulfhydryl group quantification based on molar absorptivity using Ellman’s reagent with initial substrate concentration of 3.735 mM and LuxS concentration of 69μM. Then measuring a spectrum each 10 seconds. Therefore, we plotted all gathered spectra and analyzed the 2-nitro-5-thiobenzoate (TNB⁻) absorption peak (Fig 1).

Figure 1.Dependence of TNB- absorption peak intensity on reaction time. Absorbance of reaction product TNB_- at 412 nm wavelength was recalculated into a concentration according to Lambert-Beer’s equation (5). Concentration of TNB_- dependence on reaction time is depicted in Figure 2.

Thus, experimental data was fitted to (4) equation and K_m and V_max values have been calculated. K_m calculated value was 0.38 µM, whereas V_max was equal to 3.73∙10-2 µM/s. Low K_m value indicates that luxS has high affinity towards its substrate S-(5-deoxy-D-ribos-5-yl)-L-homocysteine. Future Directions: To test our enzymatic values with traditional K_m and V_max calculation methods using different substrate concentrations. Also, there is a possibility to optimize the method of AI-2 production. Lambert W function will help other teams to calculate enzymatic properties if their future project employs expensive substrates. We were able to calculate K_m and V_max with only one substrate concentration, though further proof is required.

Figure 2.Time dependence of product (TNB⁻) concentration. Black dots represent experimental data, curved line represent global fit according to equation 4.

Sequence and Features

References

1. ↑ Sun, J., Daniel, R., Wagner-Döbler, I. & Zeng, A.-P. Is autoinducer-2 a universal signal for interspecies communication: a comparative genomic and phylogenetic analysis of the synthesis and signal transduction pathways. BMC Evol Biol 4, 36 (2004).
2. ↑ ^{2.0 2.1} Shen, G., Rajan, R., Zhu, J., Bell, C. E. & Pei, D. Design and Synthesis of Substrate and Intermediate Analogue Inhibitors of S -Ribosylhomocysteinase ‡. J. Med. Chem. 49, 3003–3011 (2006).
3. ↑ ^{3.0 3.1} Pei, D. & Zhu, J. Mechanism of action of S-ribosylhomocysteinase (LuxS). Current Opinion in Chemical Biology 8, 492–497 (2004).