The Crystal Structure Analysis Of PqqF Of The Pyrroloquinoline Quinone Biosynthesis Pathway

Pyrroloquinoline quinone(PQQ)is a water soluble,heat-stable,tricyclic ortho-quinone.It serves as redox cofactor for various bacterial dehydrogenases providing unique redox-features.It belongs to the third family of ortho-quinone cofactor following the well-known pyridine nucleotide(such as NAD)and flavin-dependent(such as FAD)cofactors.The whole genome of Serratia sp.FS14 reveals that it has a pqq cluster containing pqqABCDEF.PqqF belongs to the M16 family protease.It recognizes and cleaves all four peptide bonds to cut the precursor at residues tyrosine and glutamate from PqqA.To elucidate the catalytic mechanim of PqqF in the PQQ biosynthetic pathway,the gene of pqqF was amplified by PCR and cloned into the expression vector pET28a.The recombinant PqqF was successfully expressed in the E.coli C43(DE3)and purified by immobilized metal affinity chromatography(IMAC)and gel filtration chromatography.The purified PqqF was concentrated to 10 mg/ml and then used for crystallization screening against Hampton Research and MCSG kits by vapor diffusion method at 4℃.Initially,small plate-like cluster crystals were obtained in 35%tacsimate pH 7.0 condition.The grid screening and additive screens,different temperatures and adding different concentration of reducing agent into the protein solution were applied to optimize the crystallizaiton condition.Finally,the crystals were obtained at 16℃ with the addition of 10 mM β-ME by microseeding and a complete 2.5 A diffraction dataset was collected by X-ray.As PqqF shares very low sequence identity(<20%)with solved M16 family protease structures,selenomethionine-labeled PqqF was produced to determine the phase.A 2.8 A dataset was collected for the phase determination.Crystal structure of PqqF is resolved by Se-SAD method.The crystals belong to space group C2 and contain two molecules in the asymmetric unit.Each monomer of PqqF includes four structurally similar domains.The N-terminal domain consists of domain 1 and domain 2,and the C-terminal domain is made up of domain 3 and 4.The overall structure of PqqF monomer shaped like a closed clam-shell.The active site is situated in the N-terminal domain;in contrast,the conserved residues R656 and Y663 which are important for the substrate binding are located in the C-terminal domain.PqqF-N and PqqF-C,one half of a closed clam-shell,form a chamber with base dimensions of 9408 A3 much smaller than the insulin-degrading enzyme(IDE)chamber which is about 13000 A3.PqqF-N domain and PqqF-C domain are joined by a 37-amino acid long loop which is longer than linker of IDE(33-residues).The smaller chamber and a longer loop connecting the two half clam-shell may determine the specificity for its substrate PqqA.The 23-residues from the expression plasmid were introduced into the N-terminal of PqqF when construction of the recombinant PqqF.However,only 14-residues at the N-terminal of PqqF(missing the front 9 residues(MGSSHHHHH))could be seen in the electron density.A plausible explaination is that the missing residues were cut off by PqqF in the process of crystallization.This 14 amino acid residues long coil exactly protrudes into the counterpart active site.It indicates that the cleave sites of PqqF are not limited in the peptide bonds beside glutamate and tyrosine.Unexpectedly,the free N terminal nitrogen atom and the imidazole side chain of H-13 which was introduced from the expression vector complete the zinc sphere coordinated by H48,H52 of the zinc binding motif HxxEH and E129 located in helix5.The E51 acts as a base catalyst.The Delta G and binding energy of H-13 with the zinc iron is-16,9 kcal/mol and it was lower than that of other 13-residues with PqqF(Delta G-8.2 kcal/mol,binding energy of-13.6 kcal/mol).B-factor of H-13 among the remaining 14-residues is lowest.It indicated that H-13 of the N-terminal peptide made strong contacts with PqqF monomer.The extra N-terminal peptide not only occupied the active site,but also stabilized the closed,inactive conformation.