From Data Mining of Chitinophaga sp. Genome to Enzyme Discovery of a Hyperthermophilic Metallocarboxypeptidase

Abstract

For several centuries, microorganisms and enzymes have been used for many different applications. Although many enzymes with industrial applications have already been reported, different screening technologies, methods and approaches are constantly being developed in order to allow the identification of enzymes with even more interesting applications. In our work, we have performed data mining on the Chitinophaga sp. genome, a gram-negative bacterium isolated from a bacterial consortium of sugarcane bagasse isolated from an ethanol plant. The analysis of 8 Mb allowed the identification of the chtcp gene, previously annotated as putative Cht4039. The corresponding codified enzyme, denominated as ChtCP, showed the HEXXH conserved motif of family M32 from thermostable carboxypeptidases. After expression in E. coli, the recombinant enzyme was characterized biochemically. ChtCP showed the highest activity versus benziloxicarbonil Ala-Trp at pH 7.5, suggesting a preference for hydrophobic substrates. Surprisingly, the highest activity of ChtCP observed was between 55 °C and 75 °C, and 62% activity was still displayed at 100 °C. We observed that Ca²⁺, Ba²⁺, Mn²⁺ and Mg²⁺ ions had a positive effect on the activity of ChtCP, and an increase of 30 °C in the melting temperature was observed in the presence of Co²⁺. These features together with the structure of ChtCP at 1.2 Å highlight the relevance of ChtCP for further biotechnological applications.

Keywords: Chitinophaga sp.; M32 family of peptidases; metallocarboxypeptidase.

Figure 1

Sequence analysis of ChtCP. (A): Phylogenetic analysis by the Maximum Likelihood method. A total of 38 sequences of the M32 family were retrieved from the MEROPS database and together with ChtCP (in cyan), the evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model [51]. The tree with the highest log likelihood (-18173.3211) is shown here, and the percentage of trees in which the associated taxa clustered together is localized next to the branches. This tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The sequence alignment was performed using ClustalW and the output file was used to perform the evolutionary analyses in MEGA7 [50]. The description of the sequence, microorganism of origin and MEROPS identity (between brackets) is shown for each sequence. (B): Sequence alignment of ChtCP and selected sequences from M32 family retrieved from the MEROPS database. The conserved motifs are highlighted in gray and the conserved motif of hyperthermophilic metallopeptidaes (HEXXH) in pink. The sequence alignment was built by using ClustalW and the sequence identity between ChtCP and other sequences is shown in the left hand side of the figure.

Figure 2

Functional characterization of ChtCP. (A): The pH effect on ChtCP activity. Buffers with different pH were tested: sodium citrate (pH 4.0, 4.5, 5.0, 5.5 and 6.0); tris-HCl (pH 7.0, 7.5, 8.0 and 8.5); sodium bicarbonate (pH 9.0, 9.5 and 10.0); (B): The temperature effect on ChtCP activity. A total of 1 mg of ChtCP in 100 mM tris-HCl pH 8.0 was incubated for 1 h at different temperatures and the residual activity measured after incubation with 2 mM ZAR. (C): melting temperature (Tm) to ChtCP determined via thermal shift assay, where their denaturation promote the release of Sypro orange and the fluorescence (λ_excitation = 410 nm, λ_emission = 610 nm) released is measured through a ramp of temperature from 40 to 80 °C. The experiment was performed in 50 mM tris-HCl buffer with different pH values (7.0, 8.0 and 9.0). Each condition was performed in triplicate and the data were further normalized. (D): Catalytic efficiency (kcat/K_M) of ChtCP measured against benzyloxycarbonyl Ala-Arg (ZAR), benzyloxycarbonyl Ala-Ala (ZAA), benzyloxycarbonyl Ala-Trp (ZAW) or benzyloxycarbonyl Ala- Asn (ZAN). ChtCP at the concentrations of 0.41 to 1.6 µM were tested against the substrates at the concentration range from 0.195 to 25 mM. The reaction was performed in triplicate with 50 mM hepes and a pH of 8.0 at 65 °C.

Figure 3

The effect of metal ions on ChtCP activity and melting temperature. (A): Effect of metal ions on ChtCP activity. The values were normalized to a control where no metal ions were added. (B): Melting temperature of ChtCP in the presence and absence of metal ions (Co²⁺, Cd²⁺, Mn²⁺, Mg⁺², Cr³⁺ and Zn²⁺). 0.5 mM to 5 mM of CoCl_2, CdCl₂, MnSO₄, MgSO₄, CrSO⁴ and ZnCl₂) were added to 4 μg of purified ChtCP in hepes pH 8.0. The Tm was determined via thermal shift assay, being the released fluorescence of Sypro orange (λ_excitation = 410 nm, λ_emission = 610 nm) measured by qPCR Stratagene Mx 3000P (Agilent, Santa Clara, CA, USA) system in a ramp of temperature, from 50 °C to 80 °C.

Figure 4

Crystal structure of ChtCP carboxypeptidase from Chitinophaga sp. (A): The crystal structure of the carboxypeptidase ChtCP. The protein forms a dimer in the crystals structure which is believed to be the active form. (B) The electrostatic potential surface calculated from the crystal structure of ChtCP. The surface is contoured from −5 to +5. (C): A zoomed in view of the negatively charged active site groove of ChtCP. (D): The highly conserved metal binding residues of ChtCP aligned to the closely related structures PDB: 3HOA and 3HQ2. (E): ChtCP aligned to the 5 most closely related protein structures in the PDB (5GIV, 5WVU, 1WGZ, 5WVU and 3HOA) calculated by the Dali server [72] (F): The flat interface dimer between the 2 monomers of ChtCP.

Figure 5

The opening of the mouth of the substrate binding channel in various structures of M32 Carboxypeptidases. (i) Shows the carton depiction of α-5 and α-6 (ii) shows a cross section through the surface representation. (A): PDB of ChtCP (PDB: 7A03), (B): PDB: 3HOA (r.m.s.d = 1.19 Angstrom), (C): PDB: 3HQ2 (r.m.s.d = 1.927 Angstrom).