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Abstract

The present experiment investigates the function of protein PA5198 found in Pseudomonas Aeruginosa, known as Yestervin, using a series of different bioinformatic methods. Preliminary research from the protein data bank provides an enzyme consortium number of 3.4.17.13. These findings led to the hypothesis that Yestervin acts as an LD-Carboxypeptidase in biological systems. Bioinformatic tools used to confirm the function of Yestervin are as followed: PyMol identification of active sites within Yestervin using motif comparison, BLAST/SMART BLAST sequence comparisons between Yestervin and proteins of known function, Dali global alignment comparing carbon backbone structures of known proteins and Yestervin, Pyrx autodocking identifying ligand binding to a Yestervin-like protein’s active site amino acid residues, protein purification and kinetic evaluation through PNPA enzymatic assay. Results from these bioinformatic tools indicate that Yestervin is indeed a LD-Carboxypeptidase used to cleave amide bonds between L and D amino acids occurring naturally in bacterial peptidoglycan. These findings could play an important role in treatment development of Pseudomonas Aeruginosa infections, as well as provide mechanisms for fighting antibiotic-resistant bacteria in a hospital setting. 

Introduction

The vast gap of knowledge surrounding discovered proteins and their function serves as a setback for advancing knowledge in many fields of Biology. To date, around 20% of protein in even the most well-studied organisms have no descriptions on their roles in biological systems (Wood et al. 2019). These proteins could serve to explain a multitude of mechanisms that have been previously misunderstood and could open doors to many new discoveries in the field of Biology.

 The hypothetical protein PA5198, also known as Yestervin, found in Pseudomonas Aeruginosa, is a protein of unknown function requiring further inquiry. The unknown protein has an R-free value of 0.125 with 0.2% Ramachandran outliers, a total of six unique ligands (L(+)-Tartaric Acid, Di(Hydroxyethyl)ether, Glycerol, 1,2-Ethanediol, Potassium and Sodium ions), and PDB ID labeling it protein 1ZL0. Little is known about the protein of unknown function, however, it is suggested that the protein is possibly an LD-Carboxypeptidase. The enzyme consortium number (EC 3.4.17.13), which matches that of the unknown protein, suggests that the unknown protein is a LD-Carboxypeptidase known for the ability to cleave amide bonds between L and D amino acids occurring naturally in bacterial peptidoglycan. This severing of L and D amino acids takes tetrapeptides, truncates them into tripeptides, and then to peptidoglycan building blocks for the bacteria. LD-Carboxypeptidases have been the target of antibiotics due to their importance. The sensitivity to lactam antibiotics varies widely between antibiotics of the same class but correlates with the chirality of the amino acids of the antibiotic used. The catalytic class of LD-Carboxypeptidases is unknown, being named the U61 family. In Pseudomonas aeruginosa this U61 enzyme was found to have LD-Carboxypeptidase activity. A study by Korza & Bochtler (2005) found that the Pseudomonas aeruginosa recombinant converted tetrapeptides to tripeptides and had a structure of a serine peptidase with a Ser-His-Glu catalytic triad. The Ser115 and His285 of Pseudomonas aeruginosa were ruled out as part of the enzyme as the activity was not homologous to LD-Carboxypeptidase, further narrowing down the structure of the unknown protein in question. This carboxypeptidase in question was further investigated by Xu et al. (2020) focusing on the Carbapenem Resistance in Pseudomonas aeruginosa infections. 

Carbapenem is used to treat Pseudomonas aeruginosa infections in a clinical setting, but recent resistance to Carbapenem has led to further investigation into the mechanism behind the resistance. It was found that the IdcA (PA5198) gene encoding for murein tetrapeptide carboxypeptidase negatively influences ampC expression in Pseudomonas aeruginosa. The ampC expression is an important mechanism in bacteria that helps them develop resistance to many anti-bacterial drugs. With murein tetrapeptide carboxypeptidase expression leading to decreased expression of ampC, a mechanism using carboxypeptidases to combat drug resistance in bacteria could arise (Xu et al. 2020). The resistance to Carbapenem is due to a multitude of overexpressed and under-expressed genes that produce varying amounts of proteins. One of these overexpressions that allows Carbapenem resistance is the overexpression on the chromosomal gene ampC encoding for intrinsic cephalosporinase in Pseudomonas aeruginosa. This overexpression links carboxypeptidase to Carbapenem resistance, allowing for further research into therapeutic treatments regarding the carboxypeptidase in Pseudomonas aeruginosa.

Understanding the unknown protein Yestervin in Pseudomonas aeruginosa is important because of the possible clinical implications it could have. Pseudomonas aeruginosa is the most common-gram negative bacterium found in nosocomial infections. The bacteria is responsible for 16% of nosocomial pneumonia cases, 12% hospital-acquired urinary tract infections, 8% of surgical wound infections, 10% of bloodstream infections, and 38% of ventilator-associated pneumonia deaths (Bodey et al. 1983). The bacterium is most problematic in immunocompromised patients, as it is an opportunistic infection because of the bacteria’s presence throughout the natural environment. Once in the body, the infection is fast at causing problems throughout the body, from endocarditis in the heart, to eye problems, to urinary tract infections. The bacteria produces several extracellular products responsible for the extensive bodily damage and has been resistance or poorly treated with a number of different antibiotics throughout history (Van Delden & Iglewski 1998). The Yestervin protein could be the missing link in effective therapeutic treatments for Pseudomonas aeruginosa, which is why further investigation is a necessary process.

In this experiment, the Yestervin protein (1ZL0) was investigated using a series of different structural and sequential bioinformatic databases. These databases compared the hypothetical protein’s structure and amino acid sequence to proteins of known function. From these results, it is hypothesized the Yestervin protein is acting as a LD-Carboxypeptidase within biological systems.

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