Thermostable D-amino acid decarboxylases derived from Thermotoga maritima diaminopimelate decarboxylase.

Antonija Marjanovic,Marleen Otzen,Carlos J Ramírez-Palacios,Dick B Janssen,Jeroen Drenth,Siewert-Jan Marrink,Marcelo F Masman,Eva Petersen

doi:10.1093/protein/gzab016

Abstract

Diaminopimelate decarboxylases (DAPDCs) are highly selective enzymes that catalyze the common final step in different lysine biosynthetic pathways, i.e. the conversion of meso-diaminopimelate (DAP) to L-lysine. We examined the modification of the substrate specificity of the thermostable decarboxylase from Thermotoga maritima with the aim to introduce activity with 2-aminopimelic acid (2-APA) since its decarboxylation leads to 6-aminocaproic acid (6-ACA), a building block for the synthesis of nylon-6. Structure-based mutagenesis of the distal carboxylate binding site resulted in a set of enzyme variants with new activities toward different D-amino acids. One of the mutants (E315T) had lost most of its activity toward DAP and primarily acted as a 2-APA decarboxylase. We next used computational modeling to explain the observed shift in catalytic activities of the mutants. The results suggest that predictive computational protocols can support the redesign of the catalytic properties of this class of decarboxylating PLP-dependent enzymes.

Highlights

Amino acid decarboxylases play a crucial role in various catabolic and biosynthetic processes, such as putrefaction and production of hormones and neurotransmitters (Li et al, 2012)
We examined the modification of the substrate specificity of the thermostable decarboxylase from Thermotoga maritima with the aim to introduce activity with 2-aminopimelic acid (2-APA) since its decarboxylation leads to 6-aminocaproic acid (6-ACA), a building block for the synthesis of nylon-6
To obtain a diaminopimelate decarboxylase with modified selectivity and activity with 2-APA, we chose as the starting point the diaminopimelate decarboxylase from the hyperthermophilic bacterium T. maritima (TmDAPDC) of which the structure was solved by Nakamura and coworkers (PDB 2YXX)

Summary

Introduction

Amino acid decarboxylases play a crucial role in various catabolic and biosynthetic processes, such as putrefaction and production of hormones and neurotransmitters (Li et al, 2012). Two main groups of decarboxylases act on α-amino acids, i.e. prokaryotic pyruvoyl-dependent decarboxylases and the more widespread pyridoxal 5 -phosphate (PLP)-dependent decarboxylases. Aromatic α-amino acid decarboxylases belong to fold-type I PLP enzymes. This group includes DOPA decarboxylases involved in dopamine synthesis (Giardina et al, 2011). Glutamate decarboxylases, which produce γ -aminobutyric acid (GABA), belong to the fold-type I enzymes (Fenalti et al, 2007). Most of these α-amino acid decarboxylases act on an L-stereocenter, converting L-amino acids to amines (Paiardini et al, 2017)

Methods

Results

Conclusion