Three-dimensional structures of Pseudomonas aeruginosa PvcA and PvcB, two proteins involved in the synthesis of 2-isocyano-6,7-dihydroxycoumarin

Abstract

The pvcABCD operon of Pseudomonas aeruginosa encodes four proteins (PA2254, PA2255, PA2256, and PA2257) that form a cluster that is responsible for the synthesis of a cyclized isocyano derivative of tyrosine. These proteins, which were identified originally as being responsible for a step in the maturation of the chromophore of the peptide siderophore pyoverdine, have been identified recently as belonging to a family of proteins that produce small organic isonitriles. We report that strains harboring a disruption in the pvcA or pvcB genes are able to grow in iron-depleted conditions and to produce pyoverdine. Additionally, we have determined the three-dimensional crystal structures of PvcA and PvcB. The structure of PvcA demonstrates a novel enzyme architecture that is built upon a Rossmann fold. We have analyzed the sequence conservation of enzymes within this family and identified six conserved motifs. These regions of the protein cluster around a putative active site cavity. The structure of the PvcB protein confirms it is a member of the Fe2+/alpha-ketoglutarate-dependent oxygenase family of enzymes. The active site of PvcB is compared to the structures of other family members and suggests that a conformational change to order several loops will accompany the binding of ligands.

Figure 1

Product Identified in Culture Media Overexpressing the pvc operon. A plausible catalytic scheme is identified fo the production of 2-isocyano-6,7-dihydroxycoumarin via the catalytic activities of the PvcA, PvcB, and PvcC proteins. Note that it has not been chemically identified whether the hydroxylation of the C3 position of tyrosine occurs before or after the PvcA/PvcB-dependent isocyano formation and cyclization.

Figure 2

Cell Growth and Pyoverdine Production by pvcA and pvcB Mutants. Cells containing disruptions in the pvcA and pvcB genes were grown in minimal media lacking (solid lines, filled symbols) or containing (dashed lines, open symbols) the iron chelator EDDHA. A. Cell growth was monitored by absorbance at 595 nm for cultures of wildtype cells (circles), pvcA-disrupted cells (triangles), pvcB-dirupted cells (diamonds), or cells containing a deletion of PA2412 (squares), which has previously been shown to prevent production of sufficient levels of pyoverdine. B. Pyoverdine production was monitored by absorbance at 405 nm, represented with the same symbols as in panel A. The wild-type and pvc mutant strains all grow to saturation and produce pyoverdine.

Figure 3

Representative Experimental Electron Density. Maps of the experimental data calculated with coefficients and solvent flattened phases from RESOLVE are shown in stereo representation for A. PvcA at 3.0Å and B. PvcB at 2.6Å. Both maps are contoured at 2σ. Superimposed on the two maps are the final molecular models. For PvcA, where the native and derivative proteins crystallized in different space groups, the final protein model was superimposed on the model that was built into the experimental map.

Figure 4

PvcA Structure. A. A ribbon diagram is shown for the PvcA protein. The central parallel b-sheet is shown in red while the C-terminal sheet is shown in green. The N- and C-termini are labeled, as are helices 2–9, which are shown in blue. B. A topology diagram is shown for the PvcA protein. The central sheet is shown in black, while the C-terminal sheet is shown in white. The C-terminal residue of each secondary structure element is also included to allow comparison with the ribbon diagram.

Figure 5

Sequence Alignment of PvcA Homologs. A sequence alignment of the central core of the enzymes with homology to PvcA and IsnA is shown. The alignment begins after the N-terminal α-helices of PvcA and continues through most of the C-terminal domain. Six conserved motifs are identified with boxes. Proteins included in the alignment are from L. pneumophila strain Philadelphia (gi:53627540), L. pneumophila strain Paris (gi:53749995), B. bacteriovorus strain HD100 (gi:39574163), B. thailandensis E264 (gi:83650462), B. pseudomallei 668 (gi:126224365), P. aeruginosa PvcA (gi:9948281), E. carotovora subsp. SCRI1043 (gi:49612829), S. proteamaculans 568 (gi:157320712), P. luminescens subsp. laumondii TTO1 (gi:36786138), A. hydrophila subsp. hydrophila (gi:117561303), S. hygroscopicus strain AM3672 (gi:63033837), IsnA from uncultured bacteria (gi:71726309), Frankia sp. CcI3 (gi:86568305), Dit1p from S. cerevisiae (gi:927334), P. entomophila str. L48 (gi:95109172), and V. cholerae O395 (gi:146317530).

Figure 6

Location of Conserved Structural Motifs in PvcA. The proposed phosphate binding site and active site is shown for the PvcA protein. The phosphate ion, coordinated by Arg251, Ser253, and His255, from motif V and Lys263 from the following loop, is shown in stick representation. Other well conserved side chains, including the hydrophobic residues Phe117 and Val121 from motif II, Trp236 and Trp238 from motif IV, Trp279 from motif VI, and Phe203, which immediately precedes motif III, are highlighted with stick representation. Other residues that are well-conserved within the family of enzymes include Asp113 from motif II, Ser235 from motif IV, Thr277 from motif VI, and Asp207 from motif III, which forms a network of interactions with His280 from motif VI and Lys74. Waters that are positioned within the cavity are shown as blue spheres. The main chain of motif I forms the back wall of the active site in this view and is labeled with an asterisk. The N-terminal portion of helix 7, above Phe203, is not shown for clarity.

Figure 7

Crystal Structure of PvcB. A. The ribbon diagram of PvcB is shown with yellow helices and blue strands. The protein contains a larger domain with the jelly roll motif and a smaller domain composed of helices α3–α5 and the two smaller sheets. The side chains of His110, Asp112, and His259, the three residues that interact with the metal ligand, and Arg270 are shown in stick representation. To identify the active site, the molecular model of asparagine oxygenase from S. coelicolor A3(2) (PDB: 2OG7) was superimposed on the structure of PvcB. The ligands from 2OG7 are shown in pink (the products, 3-hydroxyasparagine and succinate) and as a sphere (Fe²⁺ ion, brown). The two disordered loops of PvcB, between Ser169-Tyr173 and Val200-Ser210 are shown with dashed lines. B. The trimer structure of PvcB is shown looking down the crystallographic three-fold rotation axis. Subunit A, shown on the left, is in approximately the same orientation as the single subunit shown in panel A.