Site-directed Mutagenesis Analysis of Vibrio alginolyticus PepD

The function of Asp82 superimposed upon PepD is similar to that of Asp89 upon PepV.

Asp82 is conserved in all the active enzymes of clan MH, considered to clamp the imidazolium ring of His80. Furthermore, the Nε2 of His80 is a coordinate of Zn1, and the Nδ1 is clamped to Asp82; besides, Glu150 is direct concerned with the metal binding. As a result, we wanted to mutate Asp82 and Glu150 of PepD by site-directed mutagenesis to learn more about the functional residues importance.

The mutants were generated using a QuikChange site-directed mutagenesis kit, as described in section 2.6, and the mutant plasmids transformed into E. coli. BL21(DE3)pLysS to express the mutant proteins. Following the same purification procedure as for V.

alginolyticus wild-type PepD, the mutant PepD proteins were extracted using 20 mM Tris-HCl pH 6.8 buffer, containing 200 mM imidazole, by Ni-NTA column chromtography.

The purified wild-type and mutant PepD proteins exhibited the same molecular weight, of about 55 kDa, on SDS-PAGE (Fig. 10).

Fig. 10: SDS-PAGE (12%) of purified wild-type and mutant proteins of Asp82 and

Lane M: LMW protein marker; Lane 1: PepD wild type; Lane 2: PepD E150D mutant; Lane 3: PepD E150R mutant; Lane 4: PepD E150H mutant; Lane 5: PepD D82F mutant; Lane 6:

PepD D82V mutant; Lane 7: PepD D82G mutant; Lane 8: PepD D82H; Lane 9: PepD D82E

The activity assays of the purified wild-type and mutant PepD proteins, taking

L-carnosine as a substrate, were assessed here. The wild-type PepD catalyzed the hydrolysis of L-carnosine under standard conditions, as described in section 2.5, its activity defined as 100% (Fig. 11).

Fig. 11: Enzymatic activities of wild-type and mutant Asp82 and Glu150 PepD on L-carnosine. The activity assay of the purified wild-type and mutant PepD proteins, taking L-carnosine as a substrate, were assessed. Wild-type activity was defined as 100%.

Asp82 was substituted with Gly, Val, Phe, Tyr, His, and Glu. As expected, no activity was detected for any of the Asp82 mutants. The substitution of Glu150 with Asp retained about 70% of the maximal hydrolytic activity of the wild-type enzyme, whereas substitution of Glu150 with Arg or His completely abolished enzymatic activity.

Moreover, based upon the structure model, His219, Asn260, Arg369, and Gly435 were identified as probable substrate binding residues, which may influence the catalytic mechanisms behind hydrolysis. Therefore, we created mutant PepD for these four residues, which were investigated using alanine scanning mutagenesis (Fig. 12). Arg369Ala lost enzymatic activity for hydrolyzing L-carnosine; but the other three residues did not relinquish catalytic activity, as predicted (Fig. 13). Accordingly, we calculated enzyme kinetics of these mutant proteins to determine the Vmax, Km and kcat values as compared with the wild-type PepD (Fig. 14)(Table 4).

Fig. 12: SDS-PAGE (12%) of purified wild-type and mutant proteins of His219, Asn260, Arg369, and Gly435. Lane M: LMW protein marker; Lane 1: PepD His219Ala mutant; Lane 2: PepD Asn260Ala mutant; Lane 3: PepD Arg369Ala mutant; Lane 4: PepD Gly435Ala; Lane 5-8: Western blot analysis of purified PepD mutanted protein with anti-PepD mAbs.

Fig. 13: Enzymatic activities of wild-type and mutant His219, Asn260, Arg369, and Gly435 PepD on L-carnosine. The activity assay of the purified wild-type and mutant PepD proteins, taking L-carnosine as a substrate, were assessed here. Wild-type activity was defined as 100%.

Fig. 14: Enzyme kinetics of the mutant His219, Asn260, and Gly435 PepD proteins A Lineweaver-Burk plot, calculated from the respective Michaelis-Menten plot for mutant PepD proteins, demonstrated mutant PepD as a catalyst for the hydrolysis of

L-carnosine. Protein concentration of PepD was 1 uM, with catalytic reagents that catalyzed the hydrolysis of L-carnosine in 50 mM Tris-HCl, pH 6.8 at 37^oC.

Earlier investigations to determine kinetic has shown the apparent KM value of wild-type V. alginolyticus PepD activity on L-carnosine to be 0.36 mM. The turnover number (kcat) and catalytic efficiency (kcat/KM) of V. alginolyticus PepD were 8.6 min^-1 and 0.398 mM^-1s^-1, respectively. The mutant PepD proteins were causing the various values shifted on kinetic parameters.

Table 4: Kinetic parameters for the hydrolysis of L-carnosine using mutant V.alginolyticus PepD.

PepD Kcat(min^-1) Km(mM) Kcat/Km (mM^-1s^-1)

WT 8.6 0.36 0.398

H219A 8 0.24 0.556

N260A 6.94 0.89 0.130

G435A 4.59 0.28 0.273

3.4 Metal ion effect of PepD activity

Several metallopeptidases are known to be key players in carcinogenesis, tissue repair, neurological processes, protein maturation, hormone-level regulation, cell-cycle control and protein-degradation. The majority of co-catalytic metallohydrolases are Zn²⁺-dependent enzymes, but some require other divalent metal ion. To chang metal ion may provide information about what possible role in enzyme function. In addition, aminoacyl-histidine dipeptidase (PepD) is a 54kD metallopeptidase, which is activated by Zn²⁺ as its wild-type.

The effect of metal substitution of the Vibrio alginolyticus PepD has been investigated and described in sections 2.2, 2.7, and 2.8. The apo-PepD was identified using a His-tag-cleaved and dialyzed buffer containing EDTA to remove divalent metal ions and yield the inactive protein. All the metal ions tested, including Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺

and Cd²⁺, can activate apo-PepD, and exert some level of hydrolysis activity on L-carnosine.

The different metal-substituted derivatives of PepD exhibit different levels of activity (Fig.

15).

Fig. 15: Metal ion effect on PepD activity. The activity assays were performed at 37°C for 30 min in the presence of 20 mM HEPES buffer, pH 7.0, 2 mM L-carnosine, 10 μM of

purified enzyme, and 20 μM of the different metal salts. The activity was measured according to standard activity assay protocol. Values are expressed as relative activity, based upon setting the hydrolysis of L-carnosine at 100%.

Particularly, the Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺ and Cd²⁺ substituted derivatives of PepD exhibited higher levels of activity than the native PepD, in terms of the hydrolysis of

L-carnosine. Conversely, no enzymatic activity was detected with Fe²⁺, and the substitution of Zn²⁺ with Mg²⁺ resulted in ~70% restoration of optimal enzymatic activity.