Abstract
Lysine decarboxylase (LDC) catalyzes the decarboxylation of l-lysine to produce cadaverine, an important industrial platform chemical for bio-based polyamides. However, due to high flexibility at the pyridoxal 5-phosphate (PLP) binding site, use of the enzyme for cadaverine production requires continuous supplement of large amounts of PLP. In order to develop an LDC enzyme from Selenomonas ruminantium (SrLDC) with an enhanced affinity for PLP, we introduced an internal disulfide bond between Ala225 and Thr302 residues with a desire to retain the PLP binding site in a closed conformation. The SrLDCA225C/T302C mutant showed a yellow color and the characteristic UV/Vis absorption peaks for enzymes with bound PLP, and exhibited three-fold enhanced PLP affinity compared with the wild-type SrLDC. The mutant also exhibited a dramatically enhanced LDC activity and cadaverine conversion particularly under no or low PLP concentrations. Moreover, introduction of the disulfide bond rendered SrLDC more resistant to high pH and temperature. The formation of the introduced disulfide bond and the maintenance of the PLP binding site in the closed conformation were confirmed by determination of the crystal structure of the mutant. This study shows that disulfide bond-mediated spatial reconstitution can be a platform technology for development of enzymes with enhanced PLP affinity.
Highlights
L-lysine is an essential amino acid and industrially important material used in animal feed and food and dietary supplements
Due to the flexible pyridoxal 5-phosphate (PLP) binding site, the protein undergoes an open/closed conformational change at the PLP binding site depending on the PLP binding (Fig 1A)
Two loops located in the vicinity of the PLP binding site, the PLP stabilization loop (PS-loop) and the regulatory loop (R-loop), undergoes a significant structural movement depending on the PLP binding (Fig 1A)
Summary
L-lysine is an essential amino acid and industrially important material used in animal feed and food and dietary supplements. It can be synthesized from aspartate [1,2]. ASA is a precursor for the biosynthesis of various amino acids such as L-threonine, L-isoleucine, Lmethionine, and L-lysine. On the L-lysine biosynthetic pathway, ASA is condensed with pyruvate to generate dihydrodipicolinate (DHDP). DHDP reductase reduces DHDP to produce tetrahydrodipicolinate (THDP). Four different pathways for the biosynthesis of Llysine that branch out from THDP have been reported in bacteria [3]: the succinylase pathway, the acetylase pathway, the mDAP dehydrogenase pathway, and the recently discovered
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