Structure and function of the phenazine biosynthetic protein PhzF from Pseudomonas fluorescens Wulf Blankenfeldt*, Alexandre P. Kuzin†, Tatiana Skarina‡, Yuriy Korniyenko‡, Liang Tong†, Peter Bayer*, Petra Janning*, Linda S. Thomashow§¶, and Dmitri V. Mavrodiʈ *Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany; †Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027; ‡Ontario Cancer Institute and Department of Medical Biophysics, Northeast Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada M5G 2M9; §U.S. Department of Agriculture Agricultural Research Service, Root Disease and Biological Control Research Unit, Pullman, WA 99164-6430; and ʈDepartment of Plant Pathology, Washington State University, Pullman, WA 99164-6430 Communicated by R. James Cook, Washington State University, Pullman, WA, October 5, 2004 (received for review July 23, 2004) Phenazines produced by Pseudomonas and Streptomyces spp. are deoxyisochorismic acid (ADIC), formed by PhzE from chorismate, heterocyclic nitrogen-containing metabolites with antibiotic, anti- is converted by PhzD, an isochorismatase, to trans-2,3-dihydro-3- tumor, and antiparasitic activity. The antibiotic properties of pyo- hydroxyanthranilic acid (DHHA) (Fig. 1). Subsequently, poorly cyanin, produced by Pseudomonas aeruginosa, were recognized in characterized steps likely involving the condensation of two iden- the 1890s, although this blue phenazine is now known to be a tical DHHA molecules are then required to form the phenazine virulence factor in human disease. Despite their biological signif- ring system. These reactions are carried out by PhzF, which lacks icance, the biosynthesis of phenazines is not fully understood. Here sequence similarity to proteins of known function. Here we provide we present structural and functional studies of PhzF, an enzyme detailed structural and biochemical analyses of PhzF, its substrate, essential for phenazine synthesis in Pseudomonas spp. PhzF shares and its products, and we propose a catalytic mechanism for the topology with diaminopimelate epimerase DapF but lacks the same condensation of DHHA to PCA. PhzF exhibits significant struc- catalytic residues. The structure of PhzF in complex with its sub- tural similarity to members of the diaminopimelate epimerase strate, trans-2,3-dihydro-3-hydroxyanthranilic acid, suggests that (DapF) fold family of proteins, but differences in key active site it is an isomerase using the conserved glutamate E45 to abstract a residues suggest greater structural and functional diversity within proton from C3 of the substrate. The proton is returned to C1 of the this family than previously was appreciated. substrate after rearrangement of the double-bond system, yielding an enol that converts to the corresponding ketone. PhzF is a dimer Materials and Methods that may be bifunctional, providing a shielded cavity for ketone Protein Purification and Crystallization. The Phz enzymes of P. dimerization via double Schiff-base formation to produce the fluorescens 2-79 and YddE of E. coli were cloned into pET15b phenazine scaffold. Our proposed mechanism is supported by mass (Novagen) and expressed in E. coli. Proteins were purified by and NMR spectroscopy. The results are discussed in the context of standard procedures, including Ni2ϩ-affinity and size-exclusion related structures and protein sequences of unknown biochemical chromatography (12). Seleno-L-methionine labeling was achieved function. by suppression of methionine biosynthesis. The QuikChange II XL system (Stratagene) was used for site-directed mutagenesis. ͉ ͉ active site residues catalytic activity x-ray structure All crystallization experiments were performed at room temper- ature with the hanging-drop vapor-diffusion method. PhzF in the he phenazines include Ͼ80 heterocyclic nitrogen-containing unliganded form was crystallized as described in ref. 12 or as a Tnatural products synthesized by fluorescent Pseudomonas spp., complex with 3-hydroxyanthranilic acid (3OHAA) by incubating members of a few other bacterial genera, and thousands of chem- the protein (10 mg⅐mlϪ1) with 10 mM ligand in 24–30% (wt/vol) ically synthesized derivatives (1). Most naturally derived phenazines polyethylene glycol 4000͞0.1 M sodium citrate (pH 4.4–5.0)͞0.2 M are pigments and many exhibit broad-spectrum antibiotic activity NH4OAc. The latter crystals were soaked in mother liquor with 10 against bacteria, fungi, and parasites. Structurally simple phena- mM DHHA for 10 days to obtain the DHHA complex. Cryopro- zines such a phenazine-1-carboxylic acid (PCA) and its hydroxy and tection was achieved by washing crystals in mother liquor amended carboxamide derivatives are produced by beneficial strains of with 30% (vol͞vol) glycerol (PhzF) or 10% (vol͞vol) glycerol Pseudomonas on the roots of plants, where they have a critical role (complexes). Data were collected at 100 K at the European in suppressing fungal pathogens (2, 3) and can contribute to the Synchrotron Radiation Facility (Grenoble, France) or on a rotating ecological competence of the strains that produce them (4). The anode (PhzF–DHHA complex). Crystals of unliganded PhzF be- BIOCHEMISTRY antibiotic properties of pyocyanin, the blue phenazine synthesized long to space group P3221 with a ϭ b ϭ 56 Å, and c ϭ 156 Å and in human tissues by the opportunistic pathogen Pseudomonas hold one monomer per asymmetric unit. Complexes of PhzF aeruginosa, were first reported in the late 1890s, although interest crystallized in space group P21212 with cell parameters of a ϭ 93 Å, now focuses on its role as a virulence determinant in chronic and b ϭ 100 Å, and c ϭ 57 Å and contained one dimer in the asymmetric acute lung disease (5, 6). The antibiotic, antiparasitic, and antitumor unit. Data were reduced in XDS (13). YddE was crystallized with 50 activities of naturally occurring phenazine compounds also have mM sodium citrate (pH 5.3)͞2 M (NH ) SO ͞5% (vol/vol) 2-pro- stimulated interest in the development of synthetic analogues 4 2 4 panol. Crystals were cryoprotected with 2.4 M (NH ) SO ͞8% tailored to particular therapeutic applications, but progress has 4 2 4 (vol/vol) 2-propanol͞15% (vol/vol) ethylene glycol for data collec- been limited by the lack of efficient and generally applicable methods for the synthesis of substituted phenazines (1). The conserved seven-gene operon phzABCDEFG is responsible Abbreviations: DapF, diaminopimelate epimerase; DHHA, trans-2,3-dihydro-3-hydroxyan- for the synthesis of PCA by Pseudomonas fluorescens 2-79 and other thranilic acid; 3OHAA, 3-hydroxyanthranilic acid; PCA, phenazine-1-carboxylic acid. fluorescent Pseudomonas spp. (7–10). PhzC, PhzD, and PhzE are Data deposition: The atomic coordinates and structure factor amplitudes have been similar to enzymes of the shikimic acid pathway and, together with deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 1U1V (selenium deriva- PhzF, are absolutely required for phenazine synthesis. Using trans- tive, sulfate complex), 1U1W (3OHAA complex), and 1U1X (DHAA complex)]. formants of Escherichia coli expressing all or different subsets of ¶To whom correspondence should be addressed. E-mail: [email protected]. these genes, McDonald et al. (11) demonstrated that 2-amino-2- © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0407371101 PNAS ͉ November 23, 2004 ͉ vol. 101 ͉ no. 47 ͉ 16431–16436 Downloaded by guest on September 30, 2021 Fig. 1. Biosynthesis of PCA from chorismic acid via DHHA. Also shown is the proposed mechanism of action of PhzF. tion at 100 K on beamline X4A of the National Synchrotron Light model was automatically built in ARP͞WARP 6.0 (17). The second Source (Brookhaven, NY). The crystals belong to space group crystal form was phased by molecular replacement by using C2221 with a ϭ 125 Å, b ϭ 131 Å, and c ϭ 49 Å and hold one MOLREP (18). Models were manually corrected in O (19) and monomer per asymmetric unit. Data were reduced in DENZO͞ refined in REFMAC5 (20). SCALEPACK (14). The structure of YddE was determined from selenium multi- The structure of PhzF was determined from selenium single- wavelength anomalous dispersion data. The structure was solved wavelength anomalous dispersion (SAD) data collected at 1.7-Å with SOLVE (21) and manually traced in O. Refinement was carried resolution on ID14-EH1 (fixed wavelength, 0.934 Å) of the out in CNS (22). Data collection and refinement statistics are given European Synchrotron Radiation Facility (12). Selenium atoms in Table 1. Coordinates have been deposited in the Protein Data were located by SHELXD97 (15) and refined in SHARP (16). The Bank (23). Table 1. Data collection and refinement statistics Se-SAD͞sulfate 3OHAA DHHA YddE (E. coli) Data collection Wavelength,* Å 0.934͞E-ID14͞1 1.001͞E-ID14͞4 1.542͞Rotating anode 0.9793͞N-X4A Resolution, Å 30.0–1.7 (1.8–1.7)† 20.0–1.35 (1.45–1.35)† 20.0–1.88 (1.98–1.88)† 20.0–2.3 (2.38–2.3)† Space group P3221 P21212 P21212 C2221 Cell constants, Å a ϭ b ϭ 56.3, a ϭ 92.9, b ϭ 100.1, a ϭ 92.8, b ϭ 99.8, a ϭ 125.0, b ϭ 130.6, c ϭ 156.4 c ϭ 57.1 c ϭ 57.4 c ϭ 48.6 Cell angles, ° ␣ ϭ ␤ ϭ 90, ␥ ϭ 120 ␣ ϭ ␤ ϭ ␥ ϭ 90 ␣ ϭ ␤ ϭ ␥ ϭ 90 ␣ ϭ ␤ ϭ ␥ ϭ 90 Average redundancy 20.6 (15.3)† 4.0 (3.7)† 4.9 (4.9)† 4.3 (27)† I͞␴ 27.4 (9.8)† 11.3 (3.1)† 14.0 (5.4)† 12.6 (3.0)† Completeness, % 98.2 (91.1)† 99.7 (99.3)† 97.8 (95.7)† 89.5 (85.6)† Anom. completeness,‡ % 98.3 (90.9)† — — 96 (86)† § † †