(634d) Crystal Structure Based Rational Engineering of Tyrosine Decarboxylase for Efficient Preparation of Tyramine

Crystal structure based rational engineering of
tyrosine decarboxylase for efficient preparation of tyramine

Guochao Xu, Mingyang Jiang, 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 lining 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 display 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. The soluble
expression of LbTDC was achieved by
simple addition of glucose and maintaining at acidic condition. Tween80 was
found to be effective in prompting the reaction especially at high substrate
loading. As much as 400 mM tyrosine could be completely converted into tyramine
with a substrate to catalyst ratio of 29.0 g¡¤g^¨C1 and total turnover
number of 23300, ranking the highest record.

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.