Structural insight into a novel neutral metalloproteinase from Paenibacillus spp. BD3526: Implications for mechanisms of rapid inactivation and calcium-dependent stability.
The proteinase with milk-clotting activity (MCA) from Paenibacillus spp. BD3526 is characterized as a neutral metalloproteinase of 35kDa. However, the rapid reduction of its MCA during separation and purification leads to low enzyme recovery. The effects of metal ions, inactivation kinetics, and concentration of calcium on the enzymatic activities and thermal stability of the BD3526 metalloproteinase were investigated. In the absence of calcium, the residual activities of the BD3526 metalloproteinase sharply declined during the first three hours, and continued to slowly decrease thereafter. The activities were well fitted by a double-exponential decay model. The inactivation rates were significantly inhibited by calcium and the residual enzyme activities were maintained at more than 80% for 30d at room temperature with 50-100mM calcium. An intermolecular autoproteolytic mechanism was responsible for BD3526 metalloproteinase inactivation. The target protein band with MCA remained largely undegraded in the enzyme solution that was supplemented with 100mM of calcium, but gradually diminished over time in the absence of calcium. N-terminal amino acid sequencing showed that cleavage at the His252-Ala253peptide bond facilitated the conversion of the zymogen into the active enzyme. Sequence alignment revealed the presence of two highly conserved motifs, HEXXH and GXXNEXXSD, indicating that the enzyme belonged to the metalloproteinase family M4, also known as thermolysin-like proteinases (TLPs). Further structural analysis showed that the observed calcium-dependent stability of the BD3526 TLP may be attributable to a partly degenerated calcium-binding site, Ca1-2, and a mutant calcium-binding site, Ca3.
State Key Laboratory of Dairy Biotechnology, Shanghai Engineering Research Center of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy & Food Co., Ltd., Shanghai 200436, PR China; State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China.
Feng
F
Hang
State Key Laboratory of Dairy Biotechnology, Shanghai Engineering Research Center of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy & Food Co., Ltd., Shanghai 200436, PR China.
Qinbo
Q
Wang
State Key Laboratory of Dairy Biotechnology, Shanghai Engineering Research Center of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy & Food Co., Ltd., Shanghai 200436, PR China.
Qing
Q
Hong
State Key Laboratory of Dairy Biotechnology, Shanghai Engineering Research Center of Dairy Biotechnology, Technology Center and Dairy Research Institute of Bright Dairy & Food Co., Ltd., Shanghai 200436, PR China.
Caixia
C
Gao
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China.
Hao
H
Zhang
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China; Beijing Innovation Centre of Food Nutrition and Human Health, Beijing Technology & Business University, Beijing 100048, PR China. Electronic address: chenwei66@jiangnan.edu.cn.
Wei
W
Chen
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002118:Calcium / Q000737:chemistry*; D020134:Catalytic Domain; D002417:Cattle; D004795:Enzyme Stability; D005260:Female; D008658:Inactivation, Metabolic; D007700:Kinetics; D045726:Metalloproteases / Q000737:chemistry* / Q000302:isolation & purification; D008892:Milk / Q000737:chemistry / Q000382:microbiology*; D008958:Models, Molecular; D015233:Models, Statistical; D056507:Paenibacillus / Q000737:chemistry* / Q000201:enzymology; D011485:Protein Binding; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013997:Time Factors
D001426:Bacterial Proteins; D045726:Metalloproteases; D002118:Calcium
Autoproteolysis; Calcium; Metalloproteinase; Paenibacillus