(191cv) Rational Engineering of Tyrosine Decarboxylase for Efficient Preparation of Tyramine

Rational engineering of tyrosine decarboxylase for
effient preparation of tyramine

Guochao Xu, Haixia Zhu, Ye Ni

¹ The Key Laboratory of Industrial Biotechnology,
Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi
214122, Jiangsu, People¡¯s Republic of China

* Corresponding author: guochaoxu@jiangnan.edu.cn; yni@jiangna.edu.cn.

Tyrosine
decarboxylase is pyridoxal 5-phosphate (PLP)-dependent enzyme that is
responsible for the biosynthesis of biogenic amines and polyamines. TDC could
catalyze the decarboxylation of L-tyrosine to tyramine, which could act as an
endogenous ligand for neurotransmitters and neuromodulators in invertebrate nervous
systems.^[1] However, despite its important applications, only one
TDC structure from Methanococcus
jannaschii (PDB code: 3F9T, MjTDC) has been resolved and deposited in the
PDB database, and the key residues responsible for substrate binding and the catalytic
mechanism remain unclear.

A TDC coding gene from Lactobacillus brevis CGMCC 1.2028
(LbTDC) was identified and successfully expressed the soluble protein in E. coli BL21(DE3).^[2] LbTDC
shares only 25% sequence identity and 60% coverage with MjTDC. The holo and apo
structures of LbTDC in complex with PLP have been resoluted, containing
N-terminal (residues 7¨C105) domain, the PLP-binding (residues 106¨C462) domain,
and the small (residues 463¨C618) domain.^[3] K240 and H241 rotate to
the opposite orientation after PLP binding, and lead to conformational swing.
They are key residues in the binding and stabilization of cofactor according to
the semisaturation mutagenesis. Alanine scanning mutagenesis was performed, and
a number of residues within the loops around the putative substrate binding
pocket were selected. Analysis revealed that (1) D271, H192, H302, and N300 are
not essential but can alter the nature of catalysis, (2) H98, S101 and residues
395¨C398 displayed decreased decarboxylase activity when mutation into alanine,
(3) residues 294¨C299 are considered important for substrate specificity, (4)
S586 is a critical residue for substrate binding. The k_cat/K_m
of the S586A variant toward tyrosine is 600 s^¨C1¡¤mM^¨C1,
2.78-fold higher than the WT enzyme. Decreased steric hindrance and increased
hydrophobicity are possible reasons for the improved catalytic efficiency of
S586A. This study provided a structural basis for the further engineering and
application of TDC in the biocatalytic synthesis of tyramine.

Figure 1. Structure of LbTDC and key residues
for cofactor binding.

[1] Zhu, Z.T.; Munhall A.C.; Johnson, S.W. Neuropharmacology,
2007, 52, 1169¨C1178.

[2] Zhang, K.; Ni, Y. Protein Expres.
Purif., 2014, 94,
33¨C39.

[3] Komori, H.; Nitta, Y.;
Ueno, H.; Higuchi, Y. J. Biol. Chem., 2012, 287, 29175¨C29183.