2019 ; Vol.29-9: 1375~1382
|Author||Zhongmei Liu, Zhongyi Cheng, Shuangshuang Ye, Li Zhou, Zhemin Zhou|
|Place of duty||Key Laboratory of Industrial Biotechnology (Ministry of Education), School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, P.R. China |
|Title||Catalytic Ability Improvement of Phenylalanine Hydroxylase from Chromobacterium violaceum by N-Terminal Truncation and Proline Introduction|
J. Microbiol. Biotechnol.2019 ;
|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.|
|Key_word||Phenylalanine hydroxylase, semirational design, catalytic ability, thermostability, Chromobacterium violaceum, phenylketonuria|
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