Sugarcane polyphenol oxidase: Structural elucidation using molecular modeling and docking analyses

Abstract

Polyphenol oxidase (PPO) enzymes induce undesirable browning in fruits, vegetables, and juices like sugarcane and apple. Inactivation of PPO is crucial to mitigate this issue, achieved through methods like heat, radiation, ultrasound, pH adjustment, adsorbent addition, and inhibitors. Inhibitor selection is traditionally a time-consuming trial-and-error process. This study aimed to develop a homology model of sugarcane PPO and to streamline inhibitor selection using a rational approach, relying on knowledge of PPO's structure and active site. The research utilized homology modeling through SWISS-MODELER to predict the 3D structures of two types of sugarcane copper-based PPOs: met-PPO (Cu-Cu active site) and oxy-PPO (Cu-O-Cu active site), both composed of 235 amino acids. Model quality was assessed using Global Model Quality Estimation (GMQE) and Qualitative Model Energy Analysis (QMEAN), yielding favorable scores of 0.17 (GMQE), 0.44 ± 0.05 (QMEAN Co-Global) and z-score of –4.03. Ramachandran analysis indicated 78.8 % and 17.5 % of amino acid residues fell within the most favored and additional allowed regions, respectively. Docking analysis revealed strong affinities between m-hydroxybenzoic acid and p-hydroxybenzoic acid with binding energies of –4.40 Kcal/mol and –4.38 Kcal/mol, respectively, for met-PPO and oxy-PPO. Identified active site residues are ARG369, HIS325, GLU321, ALA361, and TRP313. This study enhances comprehension of sugarcane PPO and its active site, aiding rational inhibitor design to counteract undesired browning reactions.

Introduction

Sugarcane (Saccharum officinarum Linn.) is a well-known commercial crop belonging to the family Poaceae. Sugarcane has been primarily utilized for the manufacture of jaggery and crystallized sugars. Attempts are being made with limited success to develop and market sugarcane juice as a nutritional and health drink [1]. Sugarcane is planted all over the world due to the medicinal and economic benefits of its high-yielding harvests. This implies that processing this much sugarcane would unintentionally generate a considerable quantity of waste [2]. It is critical to emphasize that the development of technologies for cleaner energy production has been pushed by existing and inherent stresses on agricultural commodities and crop leftovers [3]. The improvement of the economic situation is facilitated by QMEANDisCo Global valorization of waste materials for sustained growth [4]. Plant species have metabolic capabilities for generation of both valuable and waste organic products [5]. Bagasse, a sugarcane-derived bio-waste, may contain organic molecules that may have potential to act as inhibitors. Globalization is increasing energy demand and causing global warming [6]. The demand for nutritious, fresh fruits and vegetables grows in tandem with increase in population, affordability, and globalization. To meet the challenge of the increased demand, preserving the quality and freshness of fruit and vegetables from farm to consumption is crucial. One of the prime concerns in deterioration of the quality is enzymatic browning. Therefore, arresting browning and investigating the underlying mechanism of browning and inhibiting is vital for the fruit and vegetable industry. [7].

The post-harvest stability of fruits, vegetables, and crops is of industrial relevance. Damaged or stored for an extended period of time fruits, vegetables, and crops in general, incur an undesired change mainly due to browning. The browning is undesirable and results in an unattractive change in look, texture, and quality, as well as a loss of commercial value. The browning events that occur in the presence of oxygen are due to the undesirable biotransformation of polyphenols catalyzed by the family of copper metalloenzymes known as polyphenol oxidases (PPOs) present in the items [8].

Plant physiologists and food scientists have been paying close attention to PPO. PPO can be generically classified as having monophenolic and/or o-diphenolic activity based on their precision, giving rise to classes tyrosinases (TYR) and catechol oxidases (CO) [9]. These enzymes are members of the copper enzyme family Type –3. This enzyme family also includes hemocyanins and laccases [10], [11]. It catalyzes the oxidation of diphenols to o-quinone, which is then converted to melanin as a rate-limiting enzyme in the enzymatic browning reaction [12].

Understanding of protein structure and protein folding are important aspects of present-day biological science [13]. The fast-growing field of genomics has led to an inevitable divergence in the number of amino acid sequences found in protein three-dimensional structures and experimentally demonstrated [14]. The conventional approach of isolation, crystallization, and followed structure determination of proteins is cumbersome, at times very expensive and time-consuming. The computational structural elucidation approaches have emerged as quicker, simpler, and cost-effective alternatives and provide working models of structures to understand folding and active site amino acid clusters. Those models are useful for the rational design of small molecules as mimetic or inhibitors. The homology modeling method employing consensus or molecular replacement techniques builds a three-dimensional model of the protein from its amino acid sequence and the known crystal structures of a number of standard proteins [15]. Computational and spectroscopic approaches have been used for investigation of PPOs. For example, investigations of inhibitory effects of 4-hydroxycinnamic acid and naringenin on polyphenol oxidase at different pH [7], molecular dynamics simulations combined with spectroscopic experiments to analyze the effect of high pressure on the conformational changes of polyphenol oxidase of A. bisporus at the molecular level, in order to explore the mechanism on activation and deactivation of PPO by High-pressure processing [16]. Multi-spectroscopic analyses and computational simulations combined techniques were used to study, this work revealed that the structural differences between sPPO and mPPO led to different inhibition mechanisms of PPOs by inhibitors at the molecular level, which could provide guidance for the selection of inhibitors in fruit and vegetable processing [17]. However, the structure of sugarcane PPO has not been established.

In this study, with the help of computational techniques, a homology model of sugarcane PPO was built, predicted its active site residues, and performed docking analysis choosing molecules similar to natural substrates. The outcome of this study could be of great value in understanding the activity of PPO, developing inhibitors to arrest the browning reaction and consequently improving product stability and shelf life.