Structural And Functional Study Of Paenibacillus Metalloproteinase B38-CAP

Angiotensin converting enzyme 2（ACE2）is a metalloprotease that depends on Zn²⁺.It plays an important role in life activities such as a receptor protein for SARS-Co V-2 infected hosts,an accessory protein for the amino acid transporter B₀AT1,a negative regulator in the renin-angiotensin system leading it to become a drug target for the treatment of several diseases.Nevertheless,the lack of rigor in substrate selection for ACE2 and the potential negative effects on the treatment of SARS-Co V-2 infection have limited the use of the recombinant protein ACE2 as a drug in the clinical treatment of diseases.Recently,it has been shown that B38-CAP,a member of the metalloproteinase M32 family,has a high degree of enzymatic isoformity with ACE2.It also demonstrates a good role in the treatment of hypertension,pathological cardiac hypertrophy and myocardial fibrosis,causing it to become broadly potential clinical applications.In this paper,Escherichia coli BL21（DE3）was used as a recombinant expression system and various chromatographic methods were used to prepare highly pure,homogeneous and stable B38-CAP protein.On the basis of this,the crystal structure of the enzyme and inhibitor molecule was successfully obtained by molecular replacement method with a resolution of 1.56（?）through a wide range conditions of crystallization.The structural analysis showed that although the overall structure of B38-CAP and ACE2 is not conserved,but their core catalytic amino acid residues are extremely similar in spatial arrangement,which is the structural basis for the high degree of enzymatic isoformity.The results of targeted mutagenesis and in vitro luciferase activity assays indicate that the formation of stable dimer of B38-CAP in solution is closely related to its maintenance of biological activity.The experiments showed that the mutant B38-CAP in the monomeric state,although stable in nature,lost some or even all of its activity.Subsequently,further sequence and structure comparisons revealed that the catalytic mechanism is dimer-dependent to maintain catalytic activity,it may be a common catalytic mechanism for the M32 family.In addition,we also resolved the structure of a B38-CAP mutant that exists as a monomer in solution,which can simulate the transition state of the catalytic reaction for subsequent studies.In conclusion,this paper elucidates the enzymatic isoform of B38-CAP andACE2 from the perspective of protein structure.For the first time,a theory have been proposed that the formation of homodimers to maintain catalytic activity may be a conserved molecular mechanism for the metalloproteinase M32 family.These findings not only provide a structural basis to elucidating the substrate heterogeneity and the accurate catalytic mechanism of B38-CAP,but also provide a theoretical basis to further modifiying the protein for clinical treatment.