Analysis of autodegradation sites of thermolysin and enhancement of its thermostability by modifying Leu155 at an autodegradation site

The relationship between the autodegradation and thermostability of thermolysin (TLN) was studied. Four autodegradation sites in TLN were identified in the presence of Ca(2+). One of the sites was identified as Gly(154)-Leu(155), and Leu(155) was substituted with various amino acids, X = Ala, Ser, Phe, and Gly, by site-directed mutagenesis. The thermostability at 80 degrees C increased with the amino acid substitutions in the order of Ala>Phe>Ser>Gly>Leu (WT TLN). An additional autodegradation fragment that was not observed with WT TLN appeared for all mutant TLNs examined. The autodegradation site shifted from the Gly(154)-Leu(155) bond to the X(155)-Ile(156) one with the mutation at Leu(155). Furthermore, the Ile(164)-Asp(165) bond was recognized newly as an autodegradation site in the mutant TLNs for the production of AF3'.     

Grafting of a calcium-binding loop of thermolysin to Bacillus subtilis neutral protease

The surface loop which in the Bacillus subtilis neutral protease (NP) extends from amino acid residue 188 to residue 194 was replaced, by site-directed mutagenesis, with the 10-residue segment which in the homologous polypeptide chain of thermolysin (TLN) binds calcium-4 [Matthews, B. W., Weaver, L. H., & Kester, W. R. (1974) J. Biol. Chem. 249, 8030-8044]. The mutant NP was isolated to homogeneity, and its structural, functional, calcium-binding, and stability properties were investigated. Proteolytic fragmentation with Staphylococcus aureus V8 protease of mutant NP was used to isolate and analyze the protein fragment encompassing the site of mutation, unambiguously establishing the effective insertion of the new 10-residue segment. Atomic absorption measurements allowed us to demonstrate that mutant NP binds three calcium ions instead of the two ions bound to wild-type NP, showing that indeed the chain segment grafted from TLN to NP maintains its calcium-binding properties. The mutant NP showed kinetic parameters essentially similar to those of the wild-type NP with Z-Phe-Leu-Ala-OH as substrate. The enzyme inactivation of mutant vs wild-type NP was studied as a function of free [Ca2+]. It was found that mutant NP was much less stable than the wild-type NP when enzyme solutions were dialyzed at neutral pH in the presence of [Ca2+] below 10(-3) M. On the other hand, the kinetic thermal stability to irreversible inactivation of mutant NP, when measured in the presence of 0.1 M CaCl2, was found to be increased about 2-fold over that of the wild-type NP. Thus, modulation of enzyme stability by free [Ca2+] in mutant NP correlates with similar findings previously reported for thermolysin. Overall, the results obtained indicate that protein engineering experiments can be used to prepare hybrid proteins on the basis of sequence and function analysis of homologous protein molecules and show the feasibility of engineering metal ion binding sites into proteins.     

Mechanism of thermostability in thermolysin – analysis of subsite S2 mutant enzymes of thermolysin

An aromatic amino acid at position 115 (tryptophan residue; subsite S2) in thermolysin is known to be essential for proteolytic activity of thermolysin. Mutant enzymes substituted by phenylalanine (W115F) and tyrosine (W115Y) at position 115 were expressed at similar levels as the wild type (WT) enzyme in Bacillus subtilis . The thermostability of the W115Y mutant enzyme was equal to that of the WT. However, that of the W115F mutant enzyme was significantly lower than the WT. Enzymatic kcat/Km values of W115F increased to about twice those of the WT, but W115F also seemed to promote increased autodegradation compared with the WT and W115Y enzymes.     

Effects of introducing negative charges into the molecular surface of thermolysin by site-directed mutagenesis on its activity and stability

Thermolysin is remarkably activated and stabilized by neutral salts, and surface charges are suggested important in its activity and stability. The effects of introducing negative charge into the molecular surface on its activity and stability are described. Seven serine residues were selected, and each of them was changed for aspartate by site-directed mutagenesis in a thermolysin mutant. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide, the k(cat)/K(m) values of all mutants were almost similar to that of the wild-type enzyme (WT). However, those of six out of seven mutants were enhanced 17-19 times with 4 M NaCl, being slightly higher than WT. The remaining casein-hydrolyzing activities of the S53D and S65D mutants (Ser53 and Ser65 are replaced with Asp, respectively) after 30-min incubation with 10 mM CaCl(2) at 85 degrees C were 78 and 63%, being higher than those of WT (51%) and the other mutants (35-53%). S53D was stabilized with increase in the enthalpy change of activation for thermal inactivation while S65D was with decrease in the entropy change of activation. The stability of WT was enhanced by CaCl(2) and reached the level of S53D and S65D at 100 mM, suggesting that S53D and S65D might be stabilized by reinforcement of the Ca(2+)-binding structures.     

Temperature, organic solvent and pH stabilization of the neutral protease from Salinovibrio proteolyticus: significance of the structural calcium

In order to clarify the impact of Ca-binding sites (Ca1 and 2) on the conformational stability of neutral proteases (NPs), we have analyzed the thermal, pH and organic solvent stability of a NP variant, V189P/A195E/G203D/A268E (Q-mutant), from Salinovibrio proteolyticus. This mutant has shown to bind calcium more tightly than the wild-type (WT) at Ca1 and to possess Ca2. Q-mutant was resisted against autolysis, thermoinactivation and pH denaturation in a Ca-dependent manner and exhibited better activity in organic solvents compared to the WT enzyme. These results imply that Ca1 and Ca2 are important for the conformational stability of NPs.     

Ischemic dysfunction in transgenic mice expressing troponin I lacking protein kinase C phosphorylation sites

To determine the in vivo functional significance of troponin I (TnI) protein kinase C (PKC) phosphorylation sites, we created a transgenic mouse expressing mutant TnI, in which PKC phosphorylation sites at serines-43 and -45 were replaced by alanine. When we used high-perfusate calcium as a PKC activator, developed pressures in transgenic (TG) perfused hearts were similar to wild-type (WT) hearts (P = not significant, NS), though there was a 35% and 32% decrease in peak-systolic intracellular calcium (P < 0.01) and diastolic calcium (P < 0.005), respectively. The calcium transient duration was prolonged in the TG mice also (12-27%, ANOVA, P < 0.01). During global ischemia, TG hearts developed ischemic contracture to a greater extent than WT hearts (41 +/- 18 vs. 69 +/- 10 mmHg, perfusate calcium 3.5 mM, P < 0.01). In conclusion, expression of mutant TnI lacking PKC phosphorylation sites results in a marked alteration in the calcium-pressure relationship, and thus susceptibility to ischemic contracture. The reduced intracellular calcium and prolonged calcium transients suggests that a potent feedback mechanism exists between the myofilament and the processes controlling calcium homeostasis.     

The N-terminal tails of the H2A-H2B histones affect dimer structure and stability

The histone proteins of the core nucleosome are highly basic and form heterodimers in a "handshake motif." The N-terminal tails of the histones extend beyond the canonical histone fold of the hand-shake motif and are the sites of posttranslational modifications, including lysine acetylations and serine phosphorylations, which influence chromatin structure and activity as well as alter the charge state of the tails. However, it is not well understood if these modifications are signals for recruitment of other cellular factors or if the removal of net positive charge from the N-terminal tail plays a role in the overall structure of chromatin. To elucidate the effects of the N-terminal tails on the structure and stability of histones, the highly charged N-terminal tails were truncated from the H2A and H2B histones. Three mutant dimers were studied: DeltaN-H2A/WT H2B; WT H2A/DeltaN-H2B, and DeltaN-H2A/DeltaN-H2B. The CD spectra, stabilities to urea-denaturation, and the salt-dependent stabilization of the three truncated dimers were compared with those of the wild-type dimer. The data support four conclusions regarding the effects of the N-terminal tails of H2A and H2B: (1) Removal of the N-terminal tails of H2A and H2B enhance the helical structure of the mutant heterodimers. (2) Relative to the full-length WT heterodimer, the DeltaN-H2A/WT H2B dimer is destabilized, while the WT H2A/DeltaN-H2B and DeltaN-H2A/DeltaN-H2B dimers are slightly stabilized. (3) The truncated dimers exhibit decreased m values, relative to the WT dimer, supporting the hypothesis that the N-terminal tails in the isolated dimer adopt a collapsed structure. (4) Electrostatic repulsion in the N-terminal tails decreases the stability of the H2A-H2B dimer.     

Signaling Domain of Sonic Hedgehog as Cannibalistic Calcium-Regulated Zinc-Peptidase

Sonic Hedgehog (Shh) is a representative of the evolutionary closely related class of Hedgehog proteins that have essential signaling functions in animal development. The N-terminal domain (ShhN) is also assigned to the group of LAS proteins (LAS = Lysostaphin type enzymes, D-Ala-D-Ala metalloproteases, Sonic Hedgehog), of which all members harbor a structurally well-defined center; however, it is remarkable that ShhN so far is the only LAS member without proven peptidase activity. Another unique feature of ShhN in the LAS group is a double- center close to the zinc. We have studied the effect of these calcium ions on ShhN structure, dynamics, and interactions. We find that the presence of calcium has a marked impact on ShhN properties, with the two calcium ions having different effects. The more strongly bound calcium ion significantly stabilizes the overall structure. Surprisingly, the binding of the second calcium ion switches the putative catalytic center from a state similar to LAS enzymes to a state that probably is catalytically inactive. We describe in detail the mechanics of the switch, including the effect on substrate co-ordinating residues and on the putative catalytic water molecule. The properties of the putative substrate binding site suggest that ShhN could degrade other ShhN molecules, e.g. by cleavage at highly conserved glycines in ShhN. To test experimentally the stability of ShhN against autodegradation, we compare two ShhN mutants in vitro: (1) a ShhN mutant unable to bind calcium but with putative catalytic center intact, and thus, according to our hypothesis, a constitutively active peptidase, and (2) a mutant carrying additionally mutation E177A, i.e., with the putative catalytically active residue knocked out. The in vitro results are consistent with ShhN being a cannibalistic zinc-peptidase. These experiments also reveal that the peptidase activity depends on .     

The psbo1 mutant of Arabidopsis cannot efficiently use calcium in support of oxygen evolution by photosystem II

The Arabidopsis thaliana mutant psbo1 contains a point mutation in the psbO-1 gene (At5g66570) leading to the loss of expression of the PsbO-1 protein and overexpression of the PsbO-2 protein (Murakami, R., Ifuku, K., Takabayashi, A., Shikanai, T., Endo, T., and Sato, F. (2002) FEBS Lett. 523, 138-142). Previous characterization of fluorescence induction and decay kinetics by our laboratory documented defects on both the oxidizing and reducing sides of Photosystem II. Additionally, anomalous flash oxygen yield patterns indicated that the mutant contains a defective oxygen-evolving complex that appears to exhibit anomalously long-lived S(2) and S(3) oxidation states (Liu, H., Frankel, L. K., and Bricker, T. M. (2007) Biochemistry 46, 7607-7613). In this study, we have documented that the S(2) and S(3) states in psbo1 thylakoids decay very slowly. The total flash oxygen yield of the psbo1 mutant was also significantly reduced, as was its stability. Incubation of psbo1 thylakoids at high NaCl concentrations did not increase the rate of S(2) and S(3) state decay. The oxygen-evolving complexes of the mutant did, however, exhibit somewhat enhanced stability following this treatment. Incubation with CaCl(2) had a significantly more dramatic effect. Under this condition, both the S(2) and S(3) states of the mutant decayed at nearly the same rate as the wild type, and the total oxygen yield and its stability following CaCl(2) treatment were indistinguishable from that of the wild type. These results strongly suggest that the principal defect in the psbo1 mutant is an inability to effectively utilize the calcium associated with Photosystem II. We hypothesize that the PsbO-2 protein cannot effectively sequester calcium at the oxygen-evolving site.     

Consensus mutagenesis reveals that non-helical regions influence thermal stability of horseradish peroxidase

The enzyme horseradish peroxidase has many uses in biotechnology but a stabilized derivative would have even wider applicability. To enhance thermal stability, we applied consensus mutagenesis (used successfully with other proteins) to recombinant horseradish peroxidase and generated five single-site mutants. Unexpectedly, these mutations had greater effects on steady-state kinetics than on thermal stability. Only two mutants (T102A, T110V) marginally exceeded the wild type's thermal stability (4% and 10% gain in half-life at 50 degrees C respectively); the others (Q106R, Q107D, I180F) were less stable than wild type. Stability of a five-fold combination mutant matched that of Q106R, the least-stable single mutant. These results were perplexing: the Class III plant peroxidases display wide differences in thermal stability, yet the consensus mutations failed to reflect these natural variations. We examined the sequence content of Class III peroxidases to determine if there are identifiable molecular reasons for the stability differences observed. Bioinformatic analysis validated our choice of sites and mutations and generated an archetypal peroxidase sequence for comparison with extant sequences. It seems that both genetic variation and differences in protein stability are confined to non-helical regions due to the presence of a highly conserved alpha-helical structural scaffold in these enzymes.     

Contributions of aromatic pairs to the folding and stability of long-lived human γD-crystallin

Human γD-crystallin (HγD-Crys) is a highly stable protein that remains folded in the eye lens for the majority of an individual's lifetime. HγD-Crys exhibits two homologous crystallin domains, each containing two Greek key motifs with eight β-strands. Six aromatic pairs (four Tyr/Tyr, one Tyr/Phe and one Phe/Phe) are present in the β-hairpin sequences of the Greek keys. Ultraviolet damage to the aromatic residues in lens crystallins may contribute to the genesis of cataract. Mutant proteins with these aromatic residues substituted with alanines were constructed and expressed in E. coli. All mutant proteins except F115A and F117A had lower thermal stability than the WT protein. In equilibrium experiments in guanidine hydrochloride (GuHCl), all mutant proteins had lower thermodynamic stability than the WT protein. N-terminal domain (N-td) substitutions shifted the N-td transition to lower GuHCl concentration, but the C-terminal domain (C-td) transition remained unaffected. C-td substitutions led to a more cooperative unfolding/refolding process, with both the N-td and C-td transitions shifted to lower GuHCl concentration. The aromatic pairs conserved for each Greek key motif (Greek key pairs) had larger contributions to both thermal stability and thermodynamic stability than the other pairs. Aromatic-aromatic interaction was estimated as 1.5-2.0 kcal/mol. In kinetic experiments, N-td substitutions accelerated the early phase of unfolding, while C-td substitutions accelerated the late phase, suggesting independent domain unfolding. Only substitutions of the second Greek key pair of each crystallin domain slowed refolding. The second Greek keys may provide nucleation sites during the folding of the double-Greek-key crystallin domains.     

USP13 enzyme regulates Siah2 ligase stability and activity via noncatalytic ubiquitin-binding domains

The RING finger E3 ubiquitin ligase Siah2 is implicated in control of diverse cellular biological events, including MAPK signaling and hypoxia. Here we demonstrate that Siah2 is subject to regulation by the deubiquitinating enzyme USP13. Overexpression of USP13 increases Siah2 stability by attenuating its autodegradation. Consequently, the ability of Siah2 to target its substrates prolyl hydroxylase 3 and Spry2 (Sprouty2) for ubiquitin-mediated proteasomal degradation is attenuated. Conversely, inhibition of USP13 expression with corresponding shRNA decreases the stability of both Siah2 and its substrate Spry2. Thus, USP13 limits Siah2 autodegradation and its ubiquitin ligase activity against its target substrates. Strikingly, the effect of USP13 on Siah2 is not mediated by its isopeptidase activity: mutations in its ubiquitin-binding sequences positioned within the ubiquitin-specific processing protease and ubiquitin-binding domains, but not within putative catalytic sites, abolish USP13 binding to and effect on Siah2 autodegradation and targeted ubiquitination. Notably, USP13 expression is attenuated in melanoma cells maintained under hypoxia, thereby relieving Siah2 inhibition and increasing its activity under low oxygen levels. Significantly, on melanoma tissue microarray, high nuclear expression of USP13 coincided with high nuclear expression of Siah2. Overall, this study identifies a new layer of Siah2 regulation mediated by USP13 binding to ubiquitinated Siah2 protein with a concomitant inhibitory effect on its activity under normoxia.     

Presenilin 1 mediates the turnover of telencephalin in hippocampal neurons via an autophagic degradative pathway

Presenilin 1 (PS1) interacts with telencephalin (TLN) and the amyloid precursor protein via their transmembrane domain (Annaert, W.G., C. Esselens, V. Baert, C. Boeve, G. Snellings, P. Cupers, K. Craessaerts, and B. De Strooper. 2001. Neuron. 32:579-589). Here, we demonstrate that TLN is not a substrate for gamma-secretase cleavage, but displays a prolonged half-life in PS1(-/-) hippocampal neurons. TLN accumulates in intracellular structures bearing characteristics of autophagic vacuoles including the presence of Apg12p and LC3. Importantly, the TLN accumulations are suppressed by adenoviral expression of wild-type, FAD-linked and D257A mutant PS1, indicating that this phenotype is independent from gamma-secretase activity. Cathepsin D deficiency also results in the localization of TLN to autophagic vacuoles. TLN mediates the uptake of microbeads concomitant with actin and PIP2 recruitment, indicating a phagocytic origin of TLN accumulations. Absence of endosomal/lysosomal proteins suggests that the TLN-positive vacuoles fail to fuse with endosomes/lysosomes, preventing their acidification and further degradation. Collectively, PS1 deficiency affects in a gamma-secretase-independent fashion the turnover of TLN through autophagic vacuoles, most likely by an impaired capability to fuse with lysosomes.     

Cell surface and intracellular functions for ricin galactose binding.

The role of the two galactose binding sites of ricin B chain in ricin toxicity was evaluated by studying a series of ricin point mutants. Wild-type (WT) ricin and three ricin B chain point mutants having mutations in either 1) the first galactose binding domain (site 1 mutant, Met in place of Lys-40 and Gly in place of Asn-46), 2) the second galactose binding domain (site 2 mutant, Gly in place of Asn-255), or 3) both galactose binding domains (double site mutant containing all three amino acid replacements formerly stated) were expressed in Xenopus oocytes and then reassociated with recombinant ricin A chain. The different ricin B chains were mannosylated to the same extent. Cytotoxicity of these toxins was evaluated when cell entry was mediated either by galactose-containing receptors or through an alternate receptor, the mannose receptor of macrophages. WT ricin and each of the single domain mutants was able to kill Vero cells following uptake by galactose containing receptors. Lactose blocked the toxicity of each of these ricins. Site 1 and 2 mutants were 20-40 times less potent than WT ricin, and the double site mutant had no detectable cytotoxicity. WT ricin, the site 1 mutant, and the site 2 mutant also inhibited protein synthesis of mannose receptor-containing cells. Ricin can enter these cells through either a cell-surface galactose-containing receptor or through the mannose receptor. By including lactose in the cell medium, galactose-containing receptor-mediated uptake is blocked and cytotoxicity occurs solely via the mannose receptor. WT ricin, site 1, and site 2 mutants were cytotoxic to macrophages in the presence of lactose with the relative potency, WT greater than site 2 mutant greater than site 1 mutant. The double site mutant lacked cytotoxicity either in the absence or presence of lactose. Thus, even for mannose receptor-mediated toxicity of ricin, at least one galactose binding site remains necessary for cytotoxicity and two galactose binding sites further increases potency. These results are consistent with the model that the ricin B chain galactose binding activity plays a role not only in cell surface binding but also intracellularly for ricin cytotoxicity.     

Highly conserved tyrosine 37 stabilizes desensitized states and restricts calcium permeability of ATP-gated P2X3 receptor

Tyrosine 37 in the first transmembrane (TM1) domain is highly conserved in ATP-gated P2X receptors suggesting its fundamental role. We tested whether Y37 contributes to the desensitization of P2X3 receptors, which is currently not well understood. By combining electrophysiological, imaging and modeling approaches, we studied desensitization of various Y37 P2X3 mutants and potential partners of Y37. Unlike the membrane current of the WT receptor, which desensitized in seconds, Y37A mutant current did not fully desensitize even after minutes-long applications of β,γ-meATP, α,β-meATP, ATP or 2MeS-ATP. The fractional calcium current was enhanced in the Y37A mutant. Y37F did not rescue the native P2X3 phenotype indicating a role for the hydroxyl group of Y37 for the WT receptor. Homology modeling indicated I318 or I319 in TM2 as potential partners for Y37 in the receptor closed state. We tested this hypothesis by creating a permanent interaction between the two residues via disulfide bond. Whereas single Y37C, I318C and I319C mutants were functional, the double mutants Y37C-I318C and Y37C-I319C were non-functional. Using a cyclic model of receptor operation, we suggest that the conserved tyrosine 37 links TM1 to TM2 of adjacent subunit to stabilize desensitized states and restricts calcium permeability through the ion channel.     

Relative activities and stabilities of mutant Escherichia coli tryptophan synthase alpha subunits

In vitro mutagenesis of the Escherichia coli trpA gene has yielded 66 mutant tryptophan synthase alpha subunits containing single amino acid substitutions at 49 different residue sites and 29 double and triple amino acid substitutions at 16 additional sites, all within the first 121 residues of the protein. The 66 singly altered mutant alpha subunits encoded from overexpression vectors have been examined for their ability to support growth in trpA mutant host strains and for their enzymatic and stability properties in crude extracts. With the exception of mutant alpha subunits altered at catalytic residue sites Glu-49 and Asp-60, all support growth; this includes those (48 of 66) that have no enzymatic defects and those (18 of 66) that do. The majority of the enzymatically defective mutant alpha subunits have decreased capacities for substrate (indole-3-glycerol phosphate) utilization, typical of the early trpA missense mutants isolated by in vivo selection methods. These defects vary in severity from complete loss of activity for mutant alpha subunits altered at residue positions 49 and 60 to those, altered elsewhere, that are partially (up to 40 to 50%) defective. The complete inactivation of the proteins altered at the two catalytic residue sites suggest that, as found via in vitro site-specific mutagenesis of the Salmonella typhimurium tryptophan synthetase alpha subunit, both residues probably also participate in a push-pull general acid-base catalysis of indole-3-glycerol phosphate breakdown for the E. coli enzyme as well. Other classes of mutant alpha subunits include some novel types that are defective in their functional interaction with the other tryptophan synthetase component, the beta 2 subunit. Also among the mutant alpha subunits, 19 were found altered at one or another of the 34 conserved residue sites in this portion of the alpha polypeptide sequence; surprisingly, 10 of these have wild-type enzymatic activity, and 16 of these can satisfy growth requirements of a trpA mutant host. Heat stability and potential folding-rate alterations are found in both enzymatically active and defective mutant alpha subunits. Tyr-4. Pro-28, Ser-33, Gly-44, Asp-46, Arg-89, Pro-96, and Cys-118 may be important for these properties, especially for folding. Two regions, one near Thr-24 and another near Met-101, have been also tentatively identified as important for increasing stability.     

Isolation of a single carboxyl-carboxylate proton binding site in the pore of a cyclic nucleotide-gated channel.

The pore of the catfish olfactory cyclic nucleotide-gated (CNG) channel contains four conserved glutamate residues, one from each subunit, that form a high-affinity binding site for extracellular divalent cations. Previous work showed that these residues form two independent and equivalent high-pKa (approximately 7.6) proton binding sites, giving rise to three pH-dependent conductance states, and it was suggested that the sites were formed by pairing of the glutamates into two independent carboxyl-carboxylates. To test further this physical picture, wild-type CNG subunits were coexpressed in Xenopus oocytes with subunits lacking the critical glutamate residue, and single channel currents through hybrid CNG channels containing one to three wild-type (WT) subunits were recorded. One of these hybrid channels had two pH-dependent conductance states whose occupancy was controlled by a single high-pKa protonation site. Expression of dimers of concatenated CNG channel subunits confirmed that this hybrid contained two WT and two mutant subunits, supporting the idea that a single protonation site is made from two glutamates (dimer expression also implied the subunit makeup of the other hybrid channels). Thus, the proton binding sites in the WT channel occur as a result of the pairing of two glutamate residues. This conclusion places these residues in close proximity to one another in the pore and implies that at any instant in time detailed fourfold symmetry is disrupted.     

Improving the thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 using a computationally aided method

Good protein thermostability is very important for the protein application. In this report, we propose a strategy which contained a prediction method to select residues related to protein thermal stability, but not related to protein function, and an experiment method to screen the mutants with enhanced thermostability. The prediction strategy was based on the calculated site evolutionary entropy and unfolding free energy difference between the mutant and wild-type (WT) methyl parathion hydrolase enzyme from Ochrobactrum sp. M231 [Ochr-methyl parathion hydrolase (MPH)]. As a result, seven amino acid sites within Ochr-MPH were selected and used to construct seven saturation mutagenesis libraries. The results of screening these libraries indicated that six sites could result in mutated enzymes exhibiting better thermal stability than the WT enzyme. A stepwise evolutionary approach was designed to combine these selected mutants and a mutant with four point mutations (S274Q/T183E/K197L/S192M) was selected. The T _m and T ₅₀ of the mutant enzyme were 11.7 and 10.2 °C higher, respectively, than that of the WT enzyme. The success of this design methodology for Ochr-MPH suggests that it was an efficient strategy for enhancing protein thermostability and suitable for protein engineering.