Computational and experimental study on the structure and function of phenylalanine dehydrogenase in order to increase its efficiency as a diagnosis enzyme

Phenylalanine dehydrogenase (PheDh) is used in the diagnosis of phenylketonuria (PKU). In this condition, due to the inefficiency of the enzyme phenylalanine hydroxylase, an accumulation of the amino acid phenylalanine occurs in the body, which can lead to several issues, including intellectual disability. Therefore, early diagnosis of this disease in newborns is of critical importance. For accurate disease diagnosis, the specificity of PheDh for phenylalanine and the stability of the enzyme are essential factors.

Phenylalanine dehydrogenase is a bacterial-origin enzyme belonging to the amino acid dehydrogenase family. It can use not only phenylalanine but also tyrosine (and to a lesser extent, other amino acids) as substrates. The enzyme's preference for other amino acids depends on its origin. For example, the enzyme derived from Bacillus sphaericus accepts both phenylalanine and tyrosine almost equally as substrates, while the enzyme from Bacillus badius has a natural preference for phenylalanine. Nevertheless, optimizing the enzyme’s specificity for phenylalanine and enhancing its stability could improve the quality of diagnostic testing.

In recent years, other modifications have been made to the PheDh sequences, aiming to alter the enzyme's substrate preference, and thereby produce chemical compounds that could be utilized in drug synthesis, for example. The primary objective of the present study is to introduce structural changes in the PheDh enzyme derived from Bacillus badius to enhance its efficiency (such as increasing specificity for phenylalanine and/or stability) as a diagnostic enzyme. Additionally, since the study includes both computational and experimental components, it is expected that the practical examination of theoretical results will enhance knowledge about engineering this enzyme, ultimately paving the way for further valuable modifications to this enzyme.