Crystal Structure and Function of PqqF Protein in the Pyrroloquinoline Quinone Biosynthetic Pathway

Abstract

Pyrroloquinoline quinone (PQQ) has received considerable attention due to its numerous important physiological functions. PqqA is a precursor peptide of PQQ with two conserved residues: glutamate and tyrosine. After linkage of the Cγ of glutamate and Cϵ of tyrosine by PqqE, these two residues are hypothesized to be cleaved from PqqA by PqqF. The linked glutamate and tyrosine residues are then used to synthesize PQQ. Here, we demonstrated that the pqqF gene is essential for PQQ biosynthesis as deletion of it eliminated the inhibition of prodigiosin production by glucose. We further determined the crystal structure of PqqF, which has a closed clamshell-like shape. The PqqF consists of two halves composed of an N- and a C-terminal lobe. The PqqF-N and PqqF-C lobes form a chamber with the volume of the cavity of ∼9400 Å(3) The PqqF structure conforms to the general structure of inverzincins. Compared with the most thoroughly characterized inverzincin insulin-degrading enzyme, the size of PqqF chamber is markedly smaller, which may define the specificity for its substrate PqqA. Furthermore, the 14-amino acid-residue-long tag formed by the N-terminal tag from expression vector precisely protrudes into the counterpart active site; this N-terminal tag occupies the active site and stabilizes the closed, inactive conformation. His-48, His-52, Glu-129 and His-14 from the N-terminal tag coordinate with the zinc ion. Glu-51 acts as a base catalyst. The observed histidine residue-mediated inhibition may be applicable for the design of a peptide for the inhibition of M16 metalloproteases.

Keywords: crystal structure; enzyme inhibitor; metalloprotease; quinone; structure-function.

FIGURE 1.

PQQ biosynthesis pathway. Newly formed bonds are shown in blue. R₁ and R₃ represent the N- and C-terminal portions of PqqA, respectively. This figure is modified from the Figure 2 of Ref. .

FIGURE 2.

Prodigiosin production and culture pH of wild-type FS14, FS14ΔpqqF, control, and complementary strains. A, prodigiosin content (A₅₃₄) of wild-type FS14 and FS14ΔpqqF grown in glycerol peptone broth and glycerol peptone glucose broth, respectively. B, culture pH of wild-type FS14 and FS14ΔpqqF grown in glycerol peptone broth and glycerol peptone glucose broth. C, prodigiosin content (A₅₃₄) of wild-type FS14 containing the empty vector pWDX, FS14ΔpqqF containing the empty vector pWDX, and FS14ΔpqqF containing pqqF-pWDX grown in glycerol peptone glucose broth. D, culture pH of wild-type FS14 containing the empty vector pWDX, FS14ΔpqqF containing the empty vector pWDX, and FS14ΔpqqF containing pqqF-pWDX grown in glycerol peptone glucose broth. Data are from three independent experiments. The charts show an average of three independent biological replicates per condition and genotype, and error bars indicate 1 S.D.

FIGURE 3.

Structure of PqqF. A, overall structures of PqqF dimer. The four domains of one subunit are colored green, blue, red, and magenta. The linker between domains 2 and 3 is colored cyan, and the N-terminal tag is colored yellow. The other subunit is colored gray. B, topology of secondary structural elements of PqqF monomer. The four domains of PqqF are colored according to panel A.

FIGURE 4.

Analysis of the active site of PqqF structure. A, a simulated annealing omit map (2F_o − F_c) of the active site of PqqF structure contoured at 1.0σ is shown in blue. B, details of the zinc iron coordination of PqqF structure. The His-14 of the N-terminal tag is in green, and the conserved residues Glu-122, Arg-56, and Ile-160 interacting with His-52 and His-48 are colored cyan. The conserved residues His-48, His-52, Glu-51, and Glu-129 coordinated with the zinc ion are colored yellow. C, interaction of PqqF with the N-terminal tag. All residues are shown as sticks, the residues of PqqF are colored yellow, and the residues of the N-terminal tag are colored green. The simulated annealing omit map (2F_o − F_c) of active site is contoured at 2.0σ.

FIGURE 5.

Electrostatic surface potential and the internal cavity of PqqF. A, electrostatic surface potential of the PqqF-N-terminal lobe. B, electrostatic surface potential of the PqqF-C-terminal lobe. The additional N-terminal tag is represented as green sticks, the zinc ion is represented as a gray-colored sphere, and the conserved residues His-48, His-52, Glu-129, Arg-656, and Tyr-663 around the active site are represented as yellow sticks. C, ribbon presentation of PqqF. The ∼9400 ų proteolytic chamber is shown as a gray surface representation with the zinc displayed as a purple sphere.

FIGURE 6.

Structure alignments of PqqF with IDE and E. coli pitrilysin. A, superimposed structures of PqqF with E. coli pitrilysin. The structure of E. coli pitrilysin is represented in gray, and the structure of PqqF is represented in magenta. B, superimposed structures of PqqF with IDE. The structure of IDE is represented in cyan. C, structure alignment of PqqF-N lobe with IDE- N lobe. The zinc of active center is displayed as a gray sphere, and the His-48, Glu-51, and His-52 coordinated with zinc are represented by yellow sticks. D, structure alignment of PqqF-C lobe with IDE-C lobe. The helices are presented as cylinders. The color code of panel D follows that of panels A and B. E, alignment of domain1 bearing active site of PqqF, IDE, and E. coli pitrilysin. The color code of panel E follows that of panels A and B. F, superposition of catalytically important residues of PqqF with the corresponding conserved residues of E. coli pitrilysin (gray, PDB code 1Q2L) and human IDE (cyan, PDB code 2G54). Water molecules are represented as spheres colored in red. The zinc iron is represented as a sphere colored in gray. The structures of PqqF, IDE, and E. coli pitrilysin are colored magenta, cyan, and gray, respectively. The key residues are shown using a stick representation.