Research Article: Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution

Date Published: January 17, 2017

Publisher: Public Library of Science

Author(s): Hye-Young Sagong, Kyung-Jin Kim, Eugene A. Permyakov.


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.

Partial Text

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]. The aspartate is converted into l-aspartate semialdehyde (ASA) by the consecutive reaction of two enzymes. ASA is a precursor for the biosynthesis of various amino acids such as l-threonine, l-isoleucine, l-methionine, 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). Currently, four different pathways for the biosynthesis of l-lysine 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 aminotransferase pathway [4]. Finally, DAP decarboxylase catalyzes the decarboxylation of d,l-DAP to form l-lysine.

Enhanced stability of enzymes with industrial applications may improve production yield and lead to expanded operational environments such as temperature and pH. The introduction of disulfide bonds has been used as a powerful engineering tool to increase protein stability [35,36,38]. Recent studies have also shown that disulfide bond engineering can be used in a wide range of applications such as kinetic stability and functional modification of proteins [39–41]. However, not all engineered disulfide bonds produce an improved enzyme due to the difficulty in predicting the conformation and thermodynamics of an engineered disulfide bond. Our successful protein engineering on SrLDC has two unique features compared with the previously reported works. First, we used disulfide bond-mediated spatial reconstitution at the cofactor binding site to increase cofactor affinity rather than increase the stability of protein folding itself. Second, we achieved the equivalent of killing two birds with a single stone; one engineered disulfide bond enhanced both the enzymatic activity and the resistance to pH and temperature of the target protein. In many cases, engineered disulfide bonds lead to the increase of either enzymatic activity or enzyme stability [42]. Extensive structural analysis seem to enable this protein engineering to be successful.




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