Lysine decarboxylase (LDC) is a crucial enzyme for acid stress resistance and is also utilized for the biosynthesis of cadaverine, a promising building block for bio-based polyamides. We determined the crystal structure of LDC from Selenomonas ruminantium (SrLDC). SrLDC functions as a dimer and each monomer consists of two distinct domains; a PLP-binding barrel domain and a sheet domain. We also determined the structure of SrLDC in complex with PLP and cadaverine and elucidated the binding mode of cofactor and substrate. Interestingly, compared with the apo-form of SrLDC, the SrLDC in complex with PLP and cadaverine showed a remarkable structural change at the PLP binding site. The PLP binding site of SrLDC contains the highly flexible loops with high b-factors and showed an open-closed conformational change upon the binding of PLP. In fact, SrLDC showed no LDC activity without PLP supplement, and we suggest that highly flexible PLP binding site results in low PLP affinity of SrLDC. In addition, other structurally homologous enzymes also contain the flexible PLP binding site, which indicates that high flexibility at the PLP binding site and low PLP affinity seems to be a common feature of these enzyme family.
Methionine
is an essential amino acid in all living organisms and
has been used in various industrial applications such as food and
feed additives. However, inhibition of enzymes involved in methionine
biosynthesis is considered to be a crucial bottleneck for an efficient
bio-based methionine production process. Homoserine O-succinyltransferase
fromEscherichia coli (EcHST) has been reported to be feedback inhibited by the final product
methionine. To understand the regulation mechanism of the enzyme and
generate a feedback-resistant mutant, we determined the crystal structure
of EcHST and elucidated the binding site of homoserine
and succinyl-CoA. The enzyme kinetic experiments of EcHST revealed that the enzyme is noncompetitively inhibited by methionine
with a Ki
value of 2.44 mM, and we also
identified a putative inhibitor binding site located in the vicinity
of the substrate binding site. We then generated the EcHSTT242A variant with reduced feedback inhibition with
a Ki
value of 17.40 mM.
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