Functional and Structural Analysis of a Novel Acyltransferase from Pathogenic Phytophthora melonis

Abstract

This investigation characterizes an acyltransferase enzyme responsible for the pathogenicity of Phytophthora melonis. The protein was characterized in vitro for its physicochemical properties. The biochemical characterization, including thermal and pH stability, revealed the 35 °C temperature and 7.0 pH as the optimum conditions for the enzyme. Applying the Tween-80 solution enhanced the activity up to 124.9%. Comprehensive structural annotation revealed two domains, A (ranging from residues 260 to 620) and B (ranging from 141 to 219). Domain A had transglutaminase (T-Gase) elicitor properties, while B possessed antifreeze features. Rigorous sequence characterization of the acyltransferase tagged it as a low-temperature-resistant protein. Further, the taxonomic distribution analysis of the protein highlighted three genera in Oomycetes, i.e., Pythium, Phytophthora, and Plasmopara, bearing this protein. However, some taxonomic groups other than Oomycetes (i.e., archaea and bacteria) also contained the protein. Functional studies of structurally analogous proteins spanned 10 different taxonomic groups. These revealed TGase elicitors (10%), phytopathogen effector proteins RxLR (4%), transporter family proteins (3%), and endonucleases (1%). Other analogues having one percent of their individual share were HIV tat-specific factor 1, protocadherin fat 4, transcription factor 1, and 3-hydroxyisobutyrate dehydrogenase. Because the plant infection by P. melonis is a complex process regulated by a profusion of extracellular signals secreted by both host plants and the pathogen, this study will be of help in interpreting the cross-talk in the host-pathogen system.

Figure 1

Evaluation of the enzyme activity against temperature gradient ranging from 0 to 80 °C. Temperature is plotted on the X-axis, relative activity is plotted on the Y-axis, and absolute numeric values of the enzymatic activity are labeled at respective data points on the activity curve (A). Activity retention of the enzyme acyltransferase at pH gradient 2–10. The X-axis shows pH gradient values, the Y-axis represents relative activity, and absolute numeric values of the enzymatic activity are labeled at each data point on the activity curve (B). Acyltransferase activity assay under the influence of various biochemicals representing metal salts, organic acids, and detergents. The enzyme activity was measured in terms of the formation of NBD-TG μM·min^–1·g^–1 of protein (C). Data labels show the numeric values of acyltransferase activity at each data point in terms of NBD-TG μM·min^–1·g^–1 protein.

Figure 2

Standard 3D structure of the protein H3GZF6 (A). Rotating structure of bilobed acetyltransferase with abutting residues I, V, T, and F. The amplified section is showing the adjoining residues (i.e., 538 (I), 432 (V), 558 (T), 593 (F)) of the two distinct lobes of the enzyme (B).

Figure 3

Local similarity chart of “domain A (260–620)” with the standard template of the protein. Residues have been plotted on the X-axis, while the Y-axis shows local similarity with the target residue (A). Energy distribution chart for domain A. The red star highlights the position of domain A among the energy distribution matrix (B). Local similarity chart of “domain B (141–219)” with the standard template of the protein. Residues have been plotted on the X-axis, while the Y-axis shows local similarity with the target residue (C). Energy distribution chart for domain B. The red star highlights the position of domain B among the energy distribution matrix (D).

Figure 4

Detailed structural analysis of the two domains of the protein sequence H3GZF6. Supramolecular observations were made about the two domains (i.e., domain A and domain B), and characteristics of residues were highlighted.

Figure 5

Two-dimensional structure of ligand 3-cyclohexyl-1-propylsulfonic acid (CXS) (A). Ligand molecules of CXS are attached to protein H3GZF6. A CXS ligand is interacting with six residues of chain A (i.e., E.242, T.244, M.246, F.255, Y.272, and Y.323 (B)). The second CXS ligand is hydrophobically interacting with residue V.345 (C).

Figure 6

Dendrogram showing the proteins of similar homology with H3GZF6 along with their respective genes, domains, organism names, and characterization status. The data were collected from publicly available databases, e.g., UniProt, NCBI, etc. (A). The global prevalence of the protein with the taxonomic positions of the carrying organisms. The data were collected and confirmed from the NCBI database (B). Functional details of the protein species structurally similar to H3GZF6 (ProBis algorithm) and their share in the protein pool, already reported and submitted to publicly available databases (C).