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

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 N-terminal 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 $N-{\Delta}9$ variant. Notably, the $N-{\Delta}9$ 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 $N-{\Delta}9$ 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.