EhPSAT production and oligomeric state
The purified protein was homogenous as indicated by a single protein band on SDS-PAGE (Fig. 2A) and a single peak in ESI-MS of molecular mass about 43 kDa (data not shown). The SEC profile of EhPSAT (Fig. 3A) along with glutaraldehyde crosslinking experiments (Fig. 3B) demonstrates that the recombinant EhPSAT is a homodimer under physiological conditions.
pH dependent changes in functional and structural properties of EhPSAT
Functional property
EhPSAT showed pH dependent bell shaped enzymatic activity profile for forward reaction with maximum activity at pH 8.5 (Fig. 2B). At pH 10 and 6.0, the enzyme retained about 50% and 20% residual activity, respectively.
For understanding the structural basis of such a characteristic pH dependence of enzymatic activity, structural changes in the enzyme under these conditions, were studied.
Secondary structure
The secondary structure elements are generally conserved among PSAT family of enzymes [1, 2]. The secondary structure of EhPSAT as characterized by far-UV CD shows that it is a α/β type protein (Fig. 2C inset). No significant alterations in the CD signal at 222 nm were observed for the enzyme between pH 9 and 6(Fig. 2C). Hence, pH change does not significantly affect the secondary structure of EhPSAT.
Active site microenvironment
No significant alteration in fluorescence polarization of PLP was observed between pH 6 to 9 suggesting no change in orientation of cofactor PLP of EhPSAT with change in pH (Fig. 2D). Interestingly for tryptophan residues, with decrease in pH from 10 to 6 an almost linear enhancement in fluorescence polarization was observed, (Fig. 2E). This demonstrates that low pH induces restriction in orientation of tryptophan moiety present in enzyme. EhPSAT contains 3 tryptophan residues, two in the N-terminal region and one, Trp 101, in the active site. We wanted to see whether the above observed changes correspond to the active site tryptophan moiety or not.
On reduction of PLP aldimine, the cofactor serves as a reporter molecule itself and also as an energy acceptor of tryptophan fluorescence provided the two moieties are within a distance of 5 Å [15, 16]. For EhPSAT, PLP and Trp 101 are stacked close to each other at the active site (Fig. 1B inset). Fig. 2F summarizes the effect of pH on the fluorescence spectra of EhPSAT on excitation at 295 nm. At pH 8.0 and 9.0, single fluorescence emission maxima at 335 nm and 340 nm, respectively was observed. However, at pH 6 and 7, two clear emission maxima centered at 386 and 335 nm, respectively were observed. Furthermore, on decrease in pH from 9 to 6, a decrease in intensity of signal at 335 nm and a concomitant increase in intensity of signal at 386 nm were observed. These observations demonstrate that between pH 9 and 6 the two fluorophores PLP and Trp101 come closer to each other and show FRET. The FRET studies along with the PLP polarization studies (Fig. 2D to 2F) demonstrate that with change in pH, the orientation of Trp101 residue is modified such that it comes close to PLP cofactor in 3D space.
Molecular dimension, Subunit configuration and stability
On decrease in pH from 9 to 6, decrease in the hydrodynamic radii of the enzyme (Fig. 3A) was observed indicating either pH-induced dissociation of native dimer to monomer or compaction of dimeric conformation. Chemical crosslinking is a well accepted technique for studying changes in the oligomeric status of a protein under experimental conditions [16–21]. Fig. 3B shows the SDS PAGE profile of the glutaraldehyde cross linked protein samples. A single species corresponding to dimer of enzyme was observed under all the conditions studied. The stability of dimeric configuration over a broad pH range has also been reported for PSAT from Bacillus circulans ssp. Alkalophilus. These studies collectively demonstrate that PSAT dimer is stable over a broad pH range.
Fig. 3C and 3D shows thermal denaturation profile of EhPSAT at pH 8.5 and 6 as monitored by the loss of CD signal at 222 and 415 nm. Superimposable far-UV and near UV CD signals demonstrate a high degree of structural cooperativity exist in EhPSAT under these conditions. However, for pH 8.5 and 6, Tm of about 67°C and 79°C, respectively were observed. These obeservations are in agreement with the ProTherm database as reported earlier for Bacillus circulans ssp. Alkalophilus[14]. Hence, decrease in pH leads to enhanced thermal stability of PSAT.
Effect of sodium halides on the enzymatic activity
Salts affect the physico-chemical properties of proteins primarily through modulation of electrostatic and hydrophobic interactions acting on the protein molecule. Fig. 4A shows the effect of 200 mM NaF, NaCl and NaBr on the enzymatic activity at pH 8.5. NaCl and NaBr inhibited the enzymatic activity in a concentration dependent manner, while NaF showed no such effect. Detailed studies with NaCl and NaF on pH induced changes in the functional activity (Fig. 4B) clearly show no significant change in the pH dependence of enzymatic activity for NaF. Interestingly, in presence of NaCl the maximum activity was found to be retained over a broader range of pH 7.5 to 8.5 and even at pH 6 about 40% residual activity was observed.
Effect of NaCl and NaF on pH induced modification at the active site and strain of internal aldimine of EhPSAT
With change in pH from 8.5 to 6 in the presence of NaCl, no effect on fluorescence polarization of PLP was observed (Fig. 2D). However, the tryptophan fluorescence polarization was found to enhance under these conditions, but to a significantly lower extent as compared to that in absence of NaCl. This demonstrates that the pH-induced change in orientation of tryptophan residue is modulated by the presence of NaCl (Fig. 2E). FRET analyses also support these observations, Fig. 4C and 4D show no significant alteration in the tryptophan microenvironment in presence of NaCl between pH 9 and 6. In contrast for NaF, FRET profile similar to that in absence of salt was observed (Fig. 2F).
The first step in the catalytic mechanism of the enzyme is transfer of a proton of charged substrate amino group to the unprotonated imine nitrogen of the internal Shiff's base in the Michaelis complex. This is followed by nucleophilic attack of the substrate amino group on C4' carbon atom of the cofactor resulting in formation of external aldimine intermediate between PLP and substrate. In PLP catalysis, the electron sink property of cofactor plays an important role. It has been proposed that in the catalytic mechanism the strain and distortion of conjugated π-electron system of PLP and internal aldimine in PSAT are important [22]. When the internal aldimine is protonated a single positive CD band is observed at 410-415 nm but it is shifted to 345-350 nm when the internal aldimine is deprotonated [22]. The additional file 2 shows the pH dependence of near-UV CD spectra of EhPSAT in presence and absence of NaCl. Under both conditions, between pH 6 and 8, a major CD band at 410-415 nm was observed suggesting that the protein is predominantly in protonated state under these conditions. However, for protein samples at pH 9 and 10 the major CD band is shifted to 345-350 nm suggesting that the protein is predominantly in unprotonated state. These observations indicate that the pH- induced difference in the functional activity in absence and presence of NaCl is not due to modulation of pKa of the catalytic reaction.
Effect of NaCl and NAF on pH-induced compaction and thermal unfolding of EhPSAT
Fig. 4E and 4F, presents the SEC profile of EhPSAT at pH 6,7,8 and 9 in presence of 200 mM NaCl and NaF, respectively. No significant change in molecular dimension of the protein with change in pH was observed suggesting that pH induced compaction is abolished by salts. Fig. 5A to 5D, shows the thermal unfolding at pH 8.5 and 6.0, respectively in presence of 200 mM NaCl and NaF. At pH 8.5, a significant difference in Tm associated with loss of secondary structure and dissociation of PLP from enzyme was observed suggesting the thermal denaturation process in presence of salts to be a noncooperative event. However, at pH 6.0 in presence of salts changes similar to that in absence of salts were observed.