The 1.8 Å Crystal Structure of PA2412, an MbtH-like Protein from the Pyoverdine Cluster of Pseudomonas aeruginosa

Abstract

Many bacteria use nonribosomal peptide synthetase (NRPS) proteins to produce peptide antibiotics and siderophores. The catalytic domains of the NRPS proteins are usually linked in large multidomain proteins. Often, additional proteins are coexpressed with NRPS proteins that modify the NRPS peptide products, ensure the availability of substrate building blocks, or play a role in the import or export of the NRPS product. Many NRPS clusters include a small protein of ϳ80 amino acids with homology to the MbtH protein of mycobactin synthesis in Mycobacteria tuberculosis; no function has been assigned to these proteins. Pseudomonas aeruginosa utilizes an NRPS cluster to synthesize the siderophore pyoverdine. The pyoverdine peptide contains a dihydroxyquinoline-based chromophore, as well as two formyl-N-hydroxyornithine residues, which are involved in iron binding. The pyoverdine cluster contains four modular NRPS enzymes and 10 -15 additional proteins that are essential for pyoverdine production. Coexpressed with the pyoverdine synthetic enzymes is a 72-amino acid MbtH-like family member designated PA2412. We have determined the three-dimensional structure of the PA2412 protein and describe here the structure and the location of conserved regions. Additionally, we have further analyzed a deletion mutant of the PA2412 protein for growth and pyoverdine production. Our results demonstrate that PA2412 is necessary for the production or secretion of pyoverdine at normal levels. The PA2412 deletion strain is able to use exogenously produced pyoverdine, showing that there is no defect in the uptake or utilization of the iron-pyoverdine complex.Iron is an essential cofactor for many proteins, playing both catalytic and structural roles (1-3). Because of the low solubility of free Fe 3ϩ , many bacteria produce siderophores that bind to iron. The iron-siderophore complex is then actively transported into the cell. Although some small molecules like citrate or salicylate are able to function as lower affinity siderophores, many specialized peptides with extremely high affinity for iron are produced nonribosomally by bacteria. These compounds are produced by nonribosomal peptide synthetases (NRPSs) 2 that exist as modular proteins with multiple catalytic domains joined in a single protein. Often expressed with NRPS genes are other genes that encode proteins involved in additional essential steps including the synthesis of building blocks, siderophore export, import of the Fe 3ϩ -siderophore complex, or the removal Fe 3ϩ from the imported siderophore (4, 5). Pseudomonas aeruginosa is a bacterial pathogen that commonly causes infections in patients with cystic fibrosis (6). The siderophore pyoverdine (Fig. 1) is synthesized by P. aeruginosa (3) and has been correlated with virulence (7). Pyoverdine contains a cyclic peptide chain as well as a chemically modified dihydroxyquinoline-based chromophore that is responsible for iron binding. The peptide backbone of pyoverdine is synthesized by four large, multi-module NRPS proteins. The ...

Gene Cluster Involved in the Biosynthesis of Griseobactin, a Catechol-Peptide Siderophore of Streptomyces sp. ATCC 700974

“…An MbtH-like protein is found in many of the gene clusters involved in the synthesis of siderophores and peptide and aminocoumarin antibiotics. Although the requirement of MbtH-like proteins for the formation or secretion of secondary metabolites in Streptomyces (31,54) and for that of pyoverdine at wild-type levels in Pseudomonas aeruginosa (17) has been shown experimentally, the function of these proteins is still unknown, because different mbtH-like genes can functionally replace each other (31,54).…”

The Intracellular Pathogen Rhodococcus equi Produces a Catecholate Siderophore Required for Saprophytic Growth

“…The iupU gene (nucleotides [nt] 2506417 to 2533221), encoding a protein of 960.3 kDa containing seven NRPS modules, is located 28 kb upstream of iupABC, encoding a siderophore uptake system (29). The close proximity to iupABC and the linkage with mbtH, a gene required for production or secretion of siderophores and frequently clustered with siderophore biosynthesis genes (11,47), indicated that the iupU gene could be involved in siderophore biosynthesis.…”

Role of MbtH-like Proteins in the Adenylation of Tyrosine during Aminocoumarin and Vancomycin Biosynthesis

“…Drake et al (5) solved the crystal structure of an MbtH-like protein from Pseudomonas aeruginosa. The protein displays a new protein fold and is shaped like a thin arrowhead, with the point of the arrow formed by the C-terminal ␣-helix.…”

“…in catalysis, regulation, transport, or protein-protein interactions. The three-dimensional structures of two MbtH-like proteins have been experimentally determined (4,5), but again this did not allow their function to be determined. The first biochemical evidence for the function of MbtH-like proteins in non-ribosomal peptide biosynthesis has been recently provided in two rapid reports by Felnagle et al (6) and Zhang et al (7).…”