Prolidase

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Introduction

Prolidase is a metallo-dependent hydrolase responsible for the hydrolysis of Xaa-Pro and Xaa-Hyp dipeptides. Its structure is very similar to methionine aminopeptidase, aminopeptidase P and creatinase, and belongs to the so-called "pita-bread" family together with these enzymes. In addition to structural similarity, the catalytic mechanism is also shared among members of this enzyme family. Prolidases exist in all kingdoms of life. Some people think that prolinase is involved in procuring nutrients and in antitoxin defense in prokaryotes, but the real physiological role of prolinase in unicellular organisms remains to be studied. The hydrolytic activity of bacterial prolinase has been used in the biotechnology industry to reduce the bitterness of cheese and as a decontaminating agent.

Two divalent cations bridged by the hydroxyl group in the active site are essential components of this enzyme. Prolinase from other organisms can accommodate a variety of metal cations, such as Fe²⁺, Zn²⁺, or Co²⁺, while the human prolinase shows strict requirements for manganese. Due to the relatively narrow substrate spectrum, scientists believe that prolinase and other pita-bread aminopeptidases can regulate cell function in addition to participating in digestion. In Homo sapiens, collagen metabolism is considered to be the main target of prolinase. It has been shown that several human diseases may be related to prolinase activity. Its loss caused by mutations in the PEPD gene is the cause of prolinase deficiency (PD), a rare genetic disease that currently has no cure. Prolidase activity is also related to neurodegenerative and psychiatric disorders, and more and more evidence show that it is also related to tumorigenesis.

Figure 1. Pictorial view of the human prolidase structure (Wilk, P.; et al. 2020)

Abundance and genetics

Pita-bread metalloproteinases exist in all the kingdoms of life, from bacteria and archaea to simple eukaryotes to complex multicellular organisms. So far, the exact function of prolinase in single unicellular organisms is still unclear. In Escherichia coli, the prolinase-deficient strains did not show significant phenotypic differences, although it is assumed that the mutant strains will show a decreased ability to use oligopeptides as a food source. In Bacillus subtilis, prolinase plays an important role in osmoregulation by releasing free proline. In Mycobacterium tuberculosis, prolinase variants play a role in antibiotic resistance. Due to its characteristics of hydrolyzing organophosphorus compounds, the protease may also have general antitoxin protection. It is believed that in higher eukaryotes, this prolinase is most likely to participate in extracellular matrix remodeling. The human prolinase gene (PEPD) is located on chromosome 19 and contains 15 exons with a molecular weight of 54kDa. Many mutations in the PEPD gene are closely related to prolinase deficiency. The highest degree of conservation can be observed in and around the active site of the enzyme, and it is predicted that the most deleterious mutations found in the PEPD gene are related to the most conserved regions.

Figure 2. Superposition of the respective crystal structures highlighting the crucial side chains as sticks (Wilk, P.; et al. 2020)

Biotechnology

Organophosphorus compounds (OP) are the main components of many chemical warfare gases and pesticides. The Chemical Weapons Convention banned the use of gas for military purposes and obliged countries to destroy weapons stockpiles. However, due to their extremely high stability, the destruction of OPs becomes difficult and expensive. Therefore, people strive to develop more environmentally friendly purification methods. A particularly effective enzyme class has been found, organophosphorus anhydrase (OPAA), and the most studied OPAA is the one from Alteromonas sp.

OPAAs share a high sequence identity (~50%) with E. coli prolinase and are also capable of hydrolyzing Xaa-Pro dipeptides. Similarly, prolinase also exhibits OPAA activity. For human prolinase, various methods such as enzyme immobilization and engineering have increased the affinity of the enzyme by 1,000,000 times and the hydrolysis rate of paraoxon by 30 times. The second area of application of prolinase activity is the food industry, especially the dairy branch. Microbial fermentation causes the breakdown of casein and leads to bitter-tasting, mainly hydrophobic peptides. Several peptidases that are specific to proline-containing peptides have been widely used in food production. Excessive proline enzymes used together with other peptidases will accelerate the cheese ripen and reduce the overall bitterness.