Structural modelling and functional analysis of Pseudomonas ACC deaminase: An in silico study

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Introduction
The growing need for sustainable farming techniques necessitates the development of innovative approaches to improve plant growth and stress tolerance without relying on harmful pesticides and chemical fertilizers [1].One potential substitute for these environmental damaging chemical compounds lies in utilizing plant-growthpromoting rhizobacteria (PGPR) [2].These beneficial bacteria, commonly present in soils, have the potential to effectively enhance plant growth and overall plant health [3].Among the key mechanisms utilized by PGPR is the activation of 1aminocyclopropane-1-carboxylate (ACC) deaminase enzyme, which subsequently regulates ethylene by metabolizing its immediate precursor, ACC [4].
Ethylene plays a vital role as an essential plant hormone that regulates the growth and development of plants, actively contributing to various developmental and physiological processes within them [5,6].
Furthermore, ethylene plays a role in plants' responses to stress conditions, including those caused by abiotic and biotic stressors [7,8].Additionally, it participates in regulating interactions between plants and microbes [9,10].The ACC deaminase (acdS) genes show positive selection in microbial symbionts related to various leguminous plants worldwide [10], highlighting the importance of ACC

Retrieval
The

Model Evaluation and Validation
The

Functional Analysis
The prediction of functionally interacting proteins of the acdS protein from P.
brassicacearum TY1210 strain was carried out using the STRING (https://stringdb.org/).This platform utilized to investigate the interactions between acdS

CATH Classification
The acdS protein from the P.

study 3
and microbes.Some rhizospheric bacteria possess the ACC deaminase (EC 4.1.99.4) enzyme, which can convert ACC into ammonia and α-ketobutyrate [11], thus regulating the production of ethylene.The activity of ACC deaminase is found in various gram-negative bacteria such as NUST Journal of Natural Sciences, Vol. 8, Issue 2, 2023 Structural modelling and functional analysis of Pseudomonas ACC deaminase: An in silico Cronobacter sakazakii, Mesorhizobium sp., Halomonas sp., Variovorax paradoxus, Pseudomonas sp.[12-14].The Pseudomonas genus has a wide range of diversity, consisting of approximately 250 species commonly found in soil and plant microbiomes worldwide, with some of them demonstrating the ability to promote plant growth [15].Due to their enhanced metabolic adaptability, biocontrol capabilities, rapid growth rate, capacity to thrive in diverse soil conditions, and direct interaction with plant hosts, various strains of Pseudomonas have proven important for the advancement of biotechnological and agricultural products [16].However, the identification of highly efficient PGPR strains within the Pseudomonas genus remains a challenging process.An effective approach for addressing this challenge is to select Pseudomonas strains capable of regulating the levels of plant hormones, particularly ethylene (a gaseous hormone) [6, 17].The ability of bacteria to modulate plant ethylene levels is largely dependent on the expression of the ACC deaminase enzyme, responsible for breaking down the non-proteinogenic amino acid ACC [18], the immediate precursor of ethylene in all higher plants.The capability to metabolize plant ACC deaminase has been observed to improve the plant growth-promoting potential of several microbial strains, including those within Pseudomonas spp.Furthermore, numerous studies have demonstrated that acdS gene is commonly present in bacteria which have close association with their host plants, including members from both the α and β-proteobacteria classes [19].Moreover, the plant microbiome, usually survive under stressful conditions.exhibits an increased abundance of bacteria containing the acdS gene, among which Pseudomonas is commonly found [20, 21].Based on the literature cited above, it is essential to identify and characterize potential ACC deaminase-producing bacteria to develop effective bio-fertilizers, enhancing their potential for promoting plant growth and their ability for protecting plants against different environmental stresses.However, there are limited studies that conducted in-silico analysis on the function and structure of ACC deaminase proteins.Therefore, this study aimed to assess the role of the ACC deaminase in different Pseudomonas species through computational modeling.The current study used various in-silico modeling techniques to assess the primary, secondary, and tertiary structures of the ACC deaminase protein, aiming to get a deeper knowledge of its function and underlying mechanism.
this study, the PSIPRED algorithm predicted the secondary structure of the acdS protein sequences obtained from the acdS gene in Pseudomonas brassicacearum TY1210 strain.The PSIPRED algorithm operates on artificial neural networks as its fundamental framework.This approach aims to predict the secondary structure of amino acid sequence by investing information obtained from evolutionarily related proteins.Moreover, the SOPMA web-based server was utilized to predict the secondary structure of the acdS protein.The outcomes from both servers were analyzed to aid in the prediction of the protein tertiary structure.Tertiary Structure Prediction Following secondary structure prediction, the tertiary structure of the P. brassicacearum TY1210 strain was predicted using I-TASSER server [28].Initially, I-TASSER employs a multiple threading technique to recognize structural templates from the Protein Data Bank (PDB).Subsequently, it refines the 3D models by re-threading them through the protein function database to gain insights into the function of the target protein.The resultant model generated by I-TASSER was further analyzed and validated.
quality of the I-TASSER model was evaluated through the SAVES SERVER (https://saves.mbi.ucla.edu/)using QMEAN and PROCHECK tools.The Saves server employed six programs to assess the model quality.The selection of the best model was based on the quality score provided by ERRAT, a method designed to identify inaccurately predicted areas within the protein structure, characterized by a random atom distribution.QMEAN evaluated both local and global quality aspects of a model focusing on crucial geometrical features within the protein structure.angles of amino acid residues in the acdS protein.The resulting Ramachandran plot, generated by PROCHECK server, facilitated an examination of the stereochemical quality inherent in the protein structure [29].
brassicacearum TY1210 strain was further analyzed through CATH classification.Within the CATH database, protein domains are hierarchically classified based on their folding patterns, sourced from protein structures submitted to the PDB.Domain identification and subsequent categorization within CATH involve a combination of manual and automatic processes [32].

Pseudomonas, two phylogenetic
trees were constructed in MEGA11 [23].One tree was based on the nucleotide sequences of the acdS gene, while the other tree used the amino acid sequences of the ACC deaminase protein from different Pseudomonas species.It was observed that P. brassicacearum TY1210 clustered closely with Pseudomonas sp.MPDS (Figure 1).This clustering was strongly supported by a high bootstrap value of 97, indicating significant acceptance within the cluster.The collected data strongly suggests a correlated expression of the acdS gene among different Pseudomonas species.

Figure 1 :study 9 Figure 2 :
Figure 1: Phylogenetic tree of acdS gene of various species and strains of Pseudomonas

Figure 3 :
Figure 3: Amino acid composition of acdS protein of various species and strains of Pseudomonas

Figure 8 :
Figure 8: STRING analysis of acdS protein of P. brassicacearum TY1210

Table 1 :
Gene and protein accession number of various species and strains of Pseudomonas

Table 2
the ACC deaminase in P. brassicacearum TY1210 was 25.30 kDa, while in other Pseudomonas species, it ranges from 36.80 to 37.01 kDa.All analyzed proteins exhibit

NUST Journal of Natural Sciences, Vol. 8, Issue 2, 2023 Structural modelling and functional analysis of Pseudomonas ACC deaminase: An in silico study 10Table 2 :
Physiochemical properties of acdS protein of various species and strains of Pseudomonas

Table 3 )
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Table 3 :
CATH classification of acdS protein of P. brassicacearum TY1210