Gene cloning, expression and homology modeling of first fibrinolytic enzyme from mushroom (Cordyceps militaris)

Priti Katrolia; Xiaolan Liu; Yiyu Zhao; Narasimha Kumar Kopparapu; Xiqun Zheng

doi:10.1016/j.ijbiomac.2019.09.212

Gene cloning, expression and homology modeling of first fibrinolytic enzyme from mushroom (Cordyceps militaris)

Int J Biol Macromol. 2020 Mar 1:146:897-906. doi: 10.1016/j.ijbiomac.2019.09.212. Epub 2019 Nov 11.

Authors

Priti Katrolia¹, Xiaolan Liu², Yiyu Zhao³, Narasimha Kumar Kopparapu⁴, Xiqun Zheng⁵

Affiliations

¹ Heilongjiang Provincial Key Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006, China; College of Food Science, Southwest University, Beibei District, Chongqing 400715, China.
² Heilongjiang Provincial Key Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006, China. Electronic address: liuxiaolan001@126.com.
³ Heilongjiang Provincial Key Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006, China.
⁴ Heilongjiang Provincial Key Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006, China; College of Food Science, Southwest University, Beibei District, Chongqing 400715, China. Electronic address: k20187005@swu.edu.cn.
⁵ Heilongjiang Provincial Key Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006, China; Heilongjiang Bayi Agricultural University, Daqing 163319, China.

PMID: 31726136
DOI: 10.1016/j.ijbiomac.2019.09.212

Abstract

Fibrinolytic enzymes are important thrombolytic agents for blood-clotting disorders like cardiovascular diseases. Availability of novel recombinant fibrinolytic enzymes can overcome the shortcomings of current thrombolytic drugs. With the objective of facilitating their cost-effective production for therapeutic applications and for gaining deeper insight into their structure-function, we have cloned and expressed the first fibrinolytic protease gene from Cordyceps militaris. Cordyceps militaris fibrinolytic enzyme (CmFE) has one open reading frame of 759 bp encoding "pre-pro-protein" of 252 amino acids. Recombinant CmFE was expressed as 28 kDa extracellular enzyme in Pichia pastoris which was capable of degrading fibrin clot. A structure homology model of CmFE was developed using urokinase-type plasminogen activator. The active site contains catalytic triad His41, Asp83, Ser177 and consensus sequence of GDSGG. The substrate binding residues are Asp (171), Gly (194) and Ser (192). Its trypsin-like specificity is determined by the critical Asp171 in S1 subsite. The "oxyanion hole" is formed by backbone amide hydrogen atoms of Gly-175 and Ser-177. CmFE contains six conserved cysteines forming three disulfide linkages. This is the first study describing cloning, expression and prediction of structure-function relationship of a mushroom fibrinolytic protease. Hence it has great relevance in application of fibrinolytic enzymes as thrombolytic agents.

Keywords: Cordyceps militaris; Fibrinolytic enzyme; Gene cloning; Homology model; Pichia pastoris; Thrombolysis.

MeSH terms

Amino Acid Sequence
Cloning, Molecular
Cordyceps / enzymology*
Fibrinolysis*
Fungal Proteins / chemistry*
Fungal Proteins / genetics*
Gene Expression*
Models, Molecular*
Structural Homology, Protein*
Structure-Activity Relationship

Substances

Fungal Proteins