Crystal Structures of Lysine-Preferred Racemases, the Non-Antibiotic Selectable Markers for Transgenic Plants

Abstract

Lysine racemase, a pyridoxal 5′-phosphate (PLP)-dependent amino acid racemase that catalyzes the interconversion of lysine enantiomers, is valuable to serve as a novel non-antibiotic selectable marker in the generation of transgenic plants. Here, we have determined the first crystal structure of a lysine racemase (Lyr) from Proteus mirabilis BCRC10725, which shows the highest activity toward lysine and weaker activity towards arginine. In addition, we establish the first broad-specificity amino acid racemase (Bar) structure from Pseudomonas putida DSM84, which presents not only the highest activity toward lysine but also remarkably broad substrate specificity. A complex structure of Bar-lysine is also established here. These structures demonstrate the similar fold of alanine racemase, which is a head-to-tail homodimer with each protomer containing an N-terminal (α/β)8 barrel and a C-terminal β-stranded domain. The active-site residues are located at the protomer interface that is a funnel-like cavity with two catalytic bases, one from each protomer, and the PLP binding site is at the bottom of this cavity. Structural comparisons, site-directed mutagenesis, kinetic, and modeling studies identify a conserved arginine and an adjacent conserved asparagine that fix the orientation of the PLP O3 atom in both structures and assist in the enzyme activity. Furthermore, side chains of two residues in α-helix 10 have been discovered to point toward the cavity and define the substrate specificity. Our results provide a structural foundation for the design of racemases with pre-determined substrate specificity and for the development of the non-antibiotic selection system in transgenic plants.

Efficient Production of Enantiopure d-Lysine from l-Lysine by a Two-Enzyme Cascade System

“…L-Lysine decarboxylase is one of the PLP fold type I enzymes, which selectively catalyze the decarboxylation of L-lysine to generate cadaverine [15,18]. The engineered E. coli ATS3 used in our previous study was constructed by overexpressing endogenous cadA gene encoding lysine decarboxylase [15,18].…”

“…The engineered E. coli ATS3 used in our previous study was constructed by overexpressing endogenous cadA gene encoding lysine decarboxylase [15,18]. Meanwhile, the ribose 5-phosphate-dependent pathway genes pdxS and pdxT from Bacillus subtilis were introduced into the engineered E. coli for de novo PLP biosynthesis [15]. As shown in Figure 3, the crude enzyme of ATS3 exhibited the highest L-lysine decarboxylation activities, and was identified as the best biocatalyst.…”

“…In this study, amino racemases from P. mirabilis BCRC10725 (LYR) and L. paracasei (AAR), which have been reported capable of racemizing L-lysine to D-lysine, were selected [8,15]. The recombinant E. coli BL21 (DE3) harboring plasmid pET-28a-LYR or pET-28a-AAR was induced with IPTG ( Figure 2a).…”

“…The activity of the whole-cell ATS3 containing lysine decarboxylase has been characterized in a previous study [15]. Here, the effects of pH, temperature, metal ion additive, and PLP content on the crude enzyme of ATS3 were investigated.…”

Cell-specific Labeling Enzymes for Analysis of Cell–Cell Communication in Continuous Co-culture

“…Structural studies suggest P. mirabilis Lyr contains a globular catalytic core (amino acids 37-407) distinct from the putative signal peptide (18). In an attempt to limit extracellular Lyr (while retaining the catalytic activity), we removed amino acids 1-36 from the enzyme (Lyr M37 ) and added a C-terminal KDEL ER retention motif (Lyr M37-KDEL ) ( Fig.…”