A second lysine-specific serine protease from Lysobacter sp. strain IB-9374.
A second lysyl endopeptidase gene (lepB) was found immediately upstream of the previously isolated lepA gene encoding a highly active lysyl endopeptidase in Lysobacter genomic DNA. The lepB gene consists of 2,034 nucleotides coding for a protein of 678 amino acids. Amino acid sequence alignment between the lepA and lepB gene products (LepA and LepB) revealed that the LepB precursor protein is composed of a prepeptide (20 amino acids [aa]), a propeptide (184 aa), a mature enzyme (274 aa), and a C-terminal extension peptide (200 aa). The mature enzyme region exhibited 72% sequence identity to its LepA counterpart and conserved all essential amino acids constituting the catalytic triad and the primary determining site for lysine specificity. The lepB gene encoding the propeptide and mature-enzyme portions was overexpressed in Escherichia coli, and the inclusion body produced generated active LepB through appropriate refolding and processing. The purified enzyme, a mature 274-aa lysine-specific endopeptidase, was less active and more sensitive to both temperature and denaturation with urea, guanidine hydrochloride, or sodium dodecyl sulfate than LepA. LepA-based modeling implies that LepB can fold into essentially the same three-dimensional structure as LepA by placing a peptide segment, composed of several inserted amino acids found only in LepB, outside the molecule and that the Tyr169 side chain occupies the site in which the indole ring of Trp169, a built-in modulator for unique peptidase functions of LepA, resides. The results suggest that LepB is an isozyme of LepA and probably has a tertiary structure quite similar to it.
Chohnan
Shigeru
S
College of Agriculture, Ibaraki University, 3-21-1 Chu-ou, Ami, Ibaraki 300-0393, Japan. masaki@mx.ibaraki.ac.jp
Shiraki
Kentaro
K
Yokota
Kiyonobu
K
Ohshima
Makoto
M
Kuroiwa
Natsuki
N
Ahmed
Kashfia
K
Masaki
Takeharu
T
Sakiyama
Fumio
F
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044166:Xanthomonadaceae / Q000201:enzymology* / Q000235:genetics
D007527:Isoenzymes; D008565:Membrane Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; C026515:type I signal peptidase