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Figure 1. Structural divergence between canonical cytoplasmic SerB and periplasmic SerB variant (Q5E5T9).
Figure 1. Structural divergence between canonical cytoplasmic SerB and periplasmic SerB variant (Q5E5T9).
Multiple sequence alignment comparing canonical cytoplasmic SerB (cSerB; UniProt Q5E7J2) and the periplasmic SerB variant (pSerB; UniProt Q5E5T9) from Aliivibrio fischeri.
The alignment highlights the principal structural differences:
Extended N-terminus in cSerB (blue) — an N-terminal cytosolic segment present in canonical SerB but absent in pSerB.
Signal peptide in pSerB (tan) — a predicted Sec-dependent signal sequence consistent with periplasmic targeting.
Additional internal segment in pSerB (green) — a discrete insertion not present in cSerB.
Corresponding gap in cSerB (outlined box) — region absent from the cytoplasmic homolog.
Despite overall conservation of catalytic core residues typical of haloacid dehalogenase (HAD) family phosphatases, these structural differences suggest evolutionary divergence associated with compartmental retargeting and possible regulatory adaptation.
Figure 2. Recombinant expression and purification of Q5E5T9 (pSerB).
Top left: SDS–PAGE analysis of recombinant expression in E. coli.
L, molecular weight ladder (35 kDa marker indicated);
U, uninduced culture;
I, IPTG-induced culture (1 mM IPTG).
A prominent band at ~37 kDa appears upon induction, consistent with the predicted molecular weight of Q5E5T9.
Top right: SDS–PAGE analysis of fractions collected from Sephadex G-100 size-exclusion chromatography performed in 1× TBS. Fractions 6–13 are shown; strongest enrichment of the ~37 kDa band is observed in fractions 8–10.
Bottom: Corresponding A280 chromatogram of the gel filtration run. The shaded region denotes the major protein-containing peak, with maximal absorbance centered at fractions 8–10 (star), consistent with enrichment observed by SDS–PAGE.
Together, these data demonstrate robust recombinant expression and reproducible purification of Q5E5T9 under non-denaturing conditions, yielding monodisperse, catalytically active protein suitable for biochemical and biophysical characterization.
Figure 3. Differential Scanning Fluorimetry (DSF) Analysis of Q5E5T9 Stability
Differential scanning fluorimetry (DSF) was performed to assess the thermal stability of purified Q5E5T9 in the absence (apo, blue) and presence of 5 mM MgCl₂ (orange). Fluorescence emission was monitored as the ratio F330/F350 during a 1 °C/min temperature ramp from 25–85 °C. The fluorescence ratio decreases as the protein unfolds, reflecting changes in the local environment of intrinsic tryptophan residues.
Melting temperatures (Tm) were determined by nonlinear regression of the fluorescence ratio data to a two-state Boltzmann sigmoidal unfolding model (see below)
where T is temperature, Tm is the midpoint of unfolding, and k reflects the slope of the transition. Fitting was performed using nonlinear least-squares regression (four-parameter logistic model).
The apo enzyme exhibited an apparent melting temperature of approximately 62 °C, whereas the Mg²⁺-saturated enzyme exhibited a reduced melting temperature of approximately 55 °C, indicating a ~7 °C decrease in thermal stability upon Mg²⁺ binding under these conditions.
This destabilization is consistent with metal-dependent conformational flexibility and supports the hypothesis that Q5E5T9 exhibits regulatory structural modulation rather than simple structural stabilization by Mg²⁺. The data further demonstrate that the enzyme is well-folded and thermally robust under physiological buffer conditions.
Figure 4. Serine-mediated inhibition of Q5E5T9 phosphoserine phosphatase activity monitored using pNPP hydrolysis.
(A) Michaelis–Menten analysis of Q5E5T9 activity using para-nitrophenyl phosphate (pNPP) as substrate in the absence (blue) and presence (red) of 1 mM L-serine. Reaction velocity was monitored as the change in absorbance at 405 nm (ΔA405/min), corresponding to formation of para-nitrophenolate. Data were fit using non-linear least squares regression to the Michaelis–Menten equation (v₀ = Vmax[S]/(Km + [S])).
(B) Reaction scheme illustrating hydrolysis of pNPP to para-nitrophenol and inorganic phosphate. The chromogenic signal arises from deprotonation of para-nitrophenol (pKa ≈ 9.0) to the para-nitrophenolate anion (λmax = 405 nm), enabling spectrophotometric monitoring of phosphatase activity.
Together, these data demonstrate that Q5E5T9 retains canonical HAD-type phosphatase activity while exhibiting serine-sensitive regulation.
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