Title

Active-site structure of D-threonine aldolase from a green alga Chlamydomonas reinhardtii

Conference Dates

September 15-19, 2019

Abstract

The unicellular green alga Chlamydomonas reinhardtii (C. reinhardtii) has served as a useful experimental system for many fundamental biological processes. We demonstrated that d-threonine has no inhibitory effect on the cell growth and d-threonine aldolase (DTA) activity exists in C. reinhardtii. DTA is a pyridoxal 5'-phophate (PLP) dependent enzyme and catalyzes cleavage of d-form of β-hydroxy amino acids into glycine and corresponding aldehydes. We have reported cloning, purification, characterization, and crystallization of DTA from C. reinhardtii (1,2). DTA could catalyze the reverse reaction to produce d-threonine and d-allo-threonine from glycine and acetaldehyde. This result shows that the DTA of C. reinhardtii has the potential for a useful enzyme catalyzing asymmetric synthesis of various d-form of β-hydroxy amino acids. The structure of the DTA dimer involves non-covalent interactions between both protomers in a head-to-tail arrangement. Each protomer comprises a PLP-binding TIM barrel domain together with a β-strand domain as same as other fold-type-III-PLP-dependent enzymes. In this study, we demonstrated a model structure of enzyme-substrate complex, predicted a catalytic residue and a substrate-binding pocket, and made and characterized mutant enzymes of active-site residues.

The d-threonine-DTA complex structure was built in silico, and a putative catalytic residue His216 was proposed. His216 was predicted to be the catalytic base that withdraws the hydrogen of β-hydroxy group of substrates. Demonstration of the model structure also predicted a presumptive pocket in which the side chain of d-allo-threonine is located. Gly212 is located at the entrance of the pocket and we predicted that replacement of Gly212 to bulky residues reduces enzymatic activity against d-allo-threonine.

Molecular manipulations were performed on the plasmid pCrDTA harboring the native DTA gene using a QuickChange II site-directed mutagenesis kit. The generated plasmids, pCrDTA_H216A and pCrDTA_H212L, were transformed into E. coli BL21 (DE3) cells. After cultivation, the recombinant cells were disrupted by ultrasonication and centrifuged. The gene products were purified to electrophoretic homogeneity from the supernatant using ammonium sulfate fractionation and DEAE-Sepharose and Q-stat column chromatographies. Activity of DTA was assayed using alcohol dehydrogenase coupling method. H216A mutant DTA exhibited no activity indicating that His216 residue is the catalytic base in the active site of DTA. Crystallization of substrate-H216A mutant enzyme is in progress. The activity against d-allo-threonine of H212L mutant DTA was reduced by approximately 90%. But thermostability of the mutant enzyme also reduced.

(1) Y. Hirato et al., Phytochemistry 135, 18-23, 2017

(2) Y. Hirato et al., Acta Crystallographica Section F: Structural Biology Communications 73, 86-89, 2017

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