Catalytic Ability Improvement of Phenylalanine Hydroxylase from Chromobacterium violaceum by N-Terminal Truncation and Proline Introduction

Abstract

Phenylalanine hydroxylase from Chromobacterium violaceum (CvPAH) is a monomeric enzyme that converts phenylalanine to tyrosine. It shares high amino acid identity and similar structure with a subunit of human phenylalanine hydroxylase that is a tetramer, resulting in the latent application in medications. In this study, semirational design was applied to CvPAH to improve the catalytic ability based on molecular dynamics simulation analyses. Four Nterminal truncated variants and one single point variant were constructed and characterized. The D267P variant showed a 2.1-fold increased thermal stability compared to the wild type, but lower specific activity was noted compared with the wild type. The specific activity of all truncated variants was a greater than 25% increase compared to the wild type, and these variants showed similar or slightly decreased thermostability with the exception of the N-Δ9 variant. Notably, the N-Δ9 variant exhibited a 1.2-fold increased specific activity, a 1.3-fold increased thermostability and considerably increased catalytic activity under the neutral environment compared with the wild type. These properties of the N-Δ9 variant could advance medical and pharmaceutical applications of CvPAH. Our findings indicate that the N-terminus might modulate substrate binding, and are directives for further modification and functional research of PAH and other enzymes.

N-terminal truncated phospholipase A1 accessory protein PlaS from Serratia marcescens alleviates inhibitory on host cell growth and enhances PlaA1 enzymatic activity

“…In recent years, N-terminal truncation technology has emerged as a cutting-edge approach with wide-ranging applications in various fields of research (Liu et al 2019 ; Li et al 2018a , b ; Zhou et al 2022 ). This technique involves removing a portion of the N-terminal domain of a protein, resulting in modified protein variants with altered properties and functionalities.…”