Structural And Functional Studies On Metalloprotease Epp And ?-Lactamase From Vibrio Sp.

Vibrio sp.is a class of curved or straight-rod Gram-negative bacteria,some of which can cause disease in farmed animals or even death in human.Pathogenic Vibrio species has many virulence factors,and extracellular protease is the main component.Vibrio species are generally resistant to?-lactams,and one of the main mechanisms is the production of?-lactamase to hydrolyze?-lactam antibiotics.In this study,the molecular mechanism of the extracellular protease EPP of Vibrio anguillarum and the?-lactamase VPBL of Vibrio parahaemolyticus were studied by means of structural biology.The extracellular protease is usually expressed in the form of a pre-pro-enzyme and then undergo multiple processing steps to mature into the active form.The metalloproteinase EmpA is an important virulence factor of V.anguillarum and its maturation requires the successful secretion and processing.The protease Epp was found to facilitate the processing of extracellular pro-EmpA into mature EmpA.It is highly similar to Vibrio cholerae metalloprotease PrtV.The full length contains signal peptide,N-terminal domain,M6 metalloprotease domain and two PKD domains?named as PKD1 and PKD2 respectively?.The PKD1 structure of PrtV has been analyzed,and Ca²⁺can regulate the maturation process by binding to PKD1 structure and controlling the flexibility of domain connection region.Therefore,the maturation of Epp may also be regulated by Ca²⁺binding to PKD1 structure,but the specific activation mechanism is completely unknown.As the soluble M6 metalloprotease domain of Epp was not obtained,we only analyzed the first PKD structure of Epp protease in this study?Epp-PKD1?and studied its specificity of Ca²⁺binding.Epp-PKD1 exists as a monomer in the solution,consisting of seven?-strands which form two?-sheets stacking with each other.One Ca²⁺is bound by the residues N3,Q4,D27,D29,D68 and a water molecule with a pentagonal bipyramidal geometry.Incubating the apo Epp-PKD1 with Ca²⁺but not Mg²⁺,Mn²⁺,or Zn²⁺,enhances the thermal and chemical stability of Epp-PKD1,indicating its specific binding to Ca²⁺.Although Epp-PKD1 and PrtV-PKD1 have high homology in sequence and structure,they are different in the oligomeric state and local structure at the Ca²⁺-binding site,indicating maturation of PrtV and Epp might be differently regulated by Ca²⁺.Our study is helpful to better understand PKD domain mediated Ca²⁺-regulation in the maturation of protease.The?-lactams are the most widely used antibiotics in the treatment of bacterial infection,with the advantages of low toxicity,low cost,broad-spectrum antibacterial activity and good clinical efficacy.However,bacteria can evolve resistance mechanisms that hamper their use.The chromosomally encoded class A?-lactamase mainly cause Vibrio parahaemolyticus resistance to the?-lactam antibiotics.Here we firstly show the crystal structure of a class A?-lactamase of V.parahaemolyticus?VPBL?.The VPBL structure adopts a typical class A?-lactamase fold consisting of two domains,one is?/?domain including five?-strands and three?-helices,and the other is?domain mainly composed of?-helices.The active site is located at the cleft between the two domains,composed of four conserved motifs ₇₀STFK₇₃,₁₃₀SDN₁₃₂,₁₆₆ExxLN₁₇₀ and ₂₃₄KTG₂₃₆.Compared to known class A?-lactamases,VPBL contains a conserved active site,but shows residue variations in the distal region of active site,which may put effect on catalytic kinetics to certain extent.The comparison with other?-lactamase and substrate complex reveals the structural basis of its substrate preference for penicillin,ampicillin,carbencillin,and inhibitor binding,supported by enzymatic activity.We propose that?-lactams combined with?-lactamase inhibitors is a potential approach for treating vibrio infection.At the same time,we conducted directional evolution research on the plasticity of this enzyme,and found that VPBL is plastic to develop resistance to the inhibitor by the residue substitution.These mutations can act synergistically to increase the inhibitor resistance.Therefore,careful monitoring of enzyme mutations is necessary to treat infection by?-lactams and?-lactamase inhibitor combination.The?-lactamase is the key to this therapy strategy,so the study of the structure and plasticity of the?-lactamase is helpful to treat vibrio infection by?-lactams and?-lactamase inhibitor combination.On the other hand,these results also provide us with structural information to design antibiotics and inhibitors for the treatment of vibrio infection.