Literature summary for 4.1.1.18 extracted from

Sagong, H.Y.; Kim, K.J.
Lysine decarboxylase with an enhanced affinity for pyridoxal 5-phosphate by disulfide bond-mediated spatial reconstitution (2017), PLoS ONE, 12, e0170163 .

Application

Application	Comment	Organism
synthesis	enzyme LDC plays a crucial role in the synthesis of cadaverine, an important industrial platform chemical. Cadaverine is utilized with a variety of applications such as the production of polyamides, polyurethanes, chelating agents, and additives	Selenomonas ruminantium

Cloned(Commentary)

Cloned (Comment)	Organism
gene Srldc, recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3)	Selenomonas ruminantium

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified SrLDCA225C/T302C mutant, hanging drop vapour diffusion method, mixing of 0.001 ml of 65 mg/ml protein in 40 mM Tris-HCl, pH 8.0, with 0.001 ml of reservoir solution containing 1.4 M ammonium sulfate, 0.1 M sodium cacodylate, pH 6.5, and 0.2 M sodium chloride, and equilibration against 0.5 ml of reservoir solution, 20°C, X-ray diffraction structure determination and analysis at 1.8 A resolution, molecular replacement using the structure of refined SrLDC as a model, model building	Selenomonas ruminantium

Crystallization (Comment)

Organism

purified SrLDCA225C/T302C mutant, hanging drop vapour diffusion method, mixing of 0.001 ml of 65 mg/ml protein in 40 mM Tris-HCl, pH 8.0, with 0.001 ml of reservoir solution containing 1.4 M ammonium sulfate, 0.1 M sodium cacodylate, pH 6.5, and 0.2 M sodium chloride, and equilibration against 0.5 ml of reservoir solution, 20°C, X-ray diffraction structure determination and analysis at 1.8 A resolution, molecular replacement using the structure of refined SrLDC as a model, model building

Selenomonas ruminantium

Protein Variants

Protein Variants	Comment	Organism
A225C/T302C	site-directed mutagenesis, 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, an internal disulfide bond between Ala225 and Thr302 residues is introduced with a desire to retain the PLP binding site in a closed conformation. The SrLDCA225C/T302C mutant shows bound PLP, and exhibits 3fold enhanced PLP affinity compared with the wild-type SrLDC. The mutant also exhibits a dramatically enhanced LDC activity and cadaverine conversion particularly under no or low PLP concentrations. Introduction of the disulfide bond renders mutant 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 are confirmed by determination of the crystal structure of the mutant. Mutant structure determination and analysis, overview. The mutant shows increased affinity for pyridoxal 5'-phosphate and increased activity compared to wild-type	Selenomonas ruminantium
K2C/G227C	site-directed mutagenesis, the mutant shows reduced affinity for pyridoxal 5'-phosphate and reduced activity compared to wild-type	Selenomonas ruminantium
additional information	disulfide bond-mediated spatial reconstitution can be a platform technology for development of enzymes with enhanced pyridoxal 5'-phosphate affinity	Selenomonas ruminantium

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	kinetic analysis of wild-type and mutant enzymes	Selenomonas ruminantium

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
L-lysine	Selenomonas ruminantium	-	cadaverine + CO2	-	?

Organism

Organism	UniProt	Comment	Textmining
Selenomonas ruminantium	O50657	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration	Selenomonas ruminantium

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
L-lysine	-	Selenomonas ruminantium	cadaverine + CO2	-	?

Subunits

Subunits	Comment	Organism
homodimer	2 * 44000, recombinant His-tagged enzyme, SDS-PAGE	Selenomonas ruminantium

Synonyms

Synonyms	Comment	Organism
LDC	-	Selenomonas ruminantium
SrLDC	-	Selenomonas ruminantium

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Selenomonas ruminantium

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6	-	assay at	Selenomonas ruminantium

Cofactor

Cofactor	Comment	Organism	Structure
pyridoxal 5'-phosphate	PLP, binding structure and binding mode, overview. The highly flexible active site contributes to the low affinity for pyridoxal 5'-phosphate in SrLDC. The cofactor affinity is increased in enzyme mutant A225C/T302C due to introduction of an artificial disulfide bond	Selenomonas ruminantium

General Information

General Information	Comment	Organism
evolution	Selenomonas ruminantium SrLDC shows much lower pyridoxal 5'-phosphate affinity than other pyridoxal 5'-phosphate-dependent enzymes. The highly flexible active site contributes to the low affinity for pyridoxal 5'-phosphate in SrLDC	Selenomonas ruminantium
additional information	due to the flexible pyridoxal 5'-phosphate binding site, the protein undergoes an open/closed conformational change at the PLP binding site depending on the pyridoxal 5'-phosphate binding. Especially, two loops located in the vicinity of the pyridoxal 5'-phosphate binding site, the pyridoxal 5'-phosphate stabilization loop (PS-loop) and the regulatory loop (R-loop), undergo a significant structural movement depending on the pyridoxal 5'-phosphate binding	Selenomonas ruminantium
physiological function	lysine decarboxylase (LDC) is an important enzyme for maintenance of pH homeostasis and the biosynthesis of cadaverine. Most of bacteria utilize acid stress-induced lysine decarboxylase in the response to the environmental acid stress	Selenomonas ruminantium