Acclimation and Compensating Metabolite Responses to UV-B Radiation in Natural and Transgenic Populus spp. Defective in Lignin Biosynthesis.

Plants have evolved to protect leaf mesophyll tissue from damage caused by UV-B radiation by producing an array of UV-absorbing secondary metabolites. Flavonoids (phenolic glycosides) and sinapate esters (hydroxycinnamates) have been implicated as UV-B protective compounds because of the accumulation in the leaf epidermis and the strong absorption in the wavelengths corresponding to UV. Environmental adaptations by plants also generate a suite of responses for protection against damage caused by UV-B radiation, with plants from high elevations or low latitudes generally displaying greater adaptation or tolerance to UV-B radiation. In an effort to explore the relationships between plant lignin levels and composition, the origin of growth elevation, and the hierarchical synthesis of UV-screening compounds, a collection of natural variants as well as transgenic Populus spp. were examined for sensitivity or acclimation to UV-B radiation under greenhouse and laboratory conditions. Noninvasive, ecophysiological measurements using epidermal transmittance and chlorophyll fluorescence as well as metabolite measurements using UPLC-MS generally revealed that the synthesis of anthocyanins, flavonoids, and lignin precursors are increased in Populus upon moderate to high UV-B treatment. However, poplar plants with genetic modifications that affect lignin biosynthesis, or natural variants with altered lignin levels and compositions, displayed complex changes in phenylpropanoid metabolites. A balance between elevated metabolic precursors to protective phenylpropanoids and increased biosynthesis of these anthocyanins, flavonoids, and lignin is proposed to play a role in the acclimation of Populus to UV-B radiation and may provide a useful tool in engineering plants as improved bioenergy feedstocks.

Multicolor plate reader fluorescence calibration.

Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells.

Structural Insights into the Inhibitory Mechanism of an Antibody against B7-H6, a Stress-Induced Cellular Ligand for the Natural Killer Cell Receptor NKp30.

Antibodies have been shown to block signaling through cell surface receptors using several mechanisms. The two most common are binding to the ligand-binding site of the receptor and, conversely, binding to the receptor-binding site of the ligand. Here, we investigated the inhibitory mechanism of an antibody (17B1.3) against human B7-H6, a stress-induced cellular ligand for the natural killer (NK) cell receptor NKp30. Binding of this antibody to B7-H6, a transmembrane protein expressed on tumor and other stressed cells, but not on normal cells, prevents NK cell activation via NKp30. We determined the crystal structure of antibody 17B1.3 in complex with the ectodomain of B7-H6 to 2.5Å resolution. Surprisingly, 17B1.3 binds to a site on B7-H6 that is completely distinct from the binding site for NKp30, such that 17B1.3 does not block the NKp30-B7-H6 interaction. We then asked whether 17B1.3 prevents signaling by binding to a putative site for B7-H6 dimerization. However, structure-based mutations designed to disrupt potential B7-H6 dimerization through this site did not diminish NKp30-mediated cell activation. We conclude that the bulky 17B1.3 antibody most likely acts by sterically interfering with close cell-cell contacts at the NK cell-target cell interface that are required for NK cell activation. A similar inhibitory mechanism may apply to other antibodies, including therapeutic antibodies that block signaling through cell surface receptors whose ligands are also cell surface proteins.

Development of a Model Protein Interaction Pair as a Benchmarking Tool for the Quantitative Analysis of 2-Site Protein-Protein Interactions.

A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar. It is demonstrated that this system can serve as a benchmarking tool for the quantitative analysis of 2-site protein-protein interactions. The protein interaction pair enables determination of precise binding constants for the barstar protein binding to 2 distinct sites on the bivalent barnase binding partner (termed binase), where the 2 binding sites were engineered to possess affinities that differed by 2 orders of magnitude. Multiple MIRG laboratories characterized the interaction using isothermal titration calorimetry (ITC), AUC, and surface plasmon resonance (SPR) methods to evaluate the feasibility of the system as a benchmarking model. Although general agreement was seen for the binding constants measured using solution-based ITC and AUC approaches, weaker affinity was seen for surface-based method SPR, with protein immobilization likely affecting affinity. An analysis of the results from multiple MIRG laboratories suggests that the bivalent barnase-barstar system is a suitable model for benchmarking new approaches for the quantitative characterization of complex biomolecular interactions.

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Structural basis for the binding specificity of human Recepteur d'Origine Nantais (RON) receptor tyrosine kinase to macrophage-stimulating protein.

Recepteur d'origine nantais (RON) receptor tyrosine kinase and its ligand, serum macrophage-stimulating protein (MSP), play important roles in inflammation, cell growth, migration, and epithelial to mesenchymal transition during tumor development. The binding of mature MSPαß (disulfide-linked α- and ß-chains) to RON ectodomain modulates receptor dimerization, followed by autophosphorylation of tyrosines in the cytoplasmic receptor kinase domains. Receptor recognition is mediated by binding of MSP ß-chain (MSPß) to the RON Sema. Here we report the structure of RON Sema-PSI-IPT1 (SPI1) domains in complex with MSPß at 3.0 Å resolution. The MSPß serine protease-like ß-barrel uses the degenerate serine protease active site to recognize blades 2, 3, and 4 of the ß-propeller fold of RON Sema. Despite the sequence homology between RON and MET receptor tyrosine kinase and between MSP and hepatocyte growth factor, it is well established that there is no cross-reactivity between the two receptor-ligand systems. Comparison of the structure of RON SPI1 in complex with MSPß and that of MET receptor tyrosine kinase Sema-PSI in complex with hepatocyte growth factor ß-chain reveals the receptor-ligand selectivity determinants. Analytical ultracentrifugation studies of the SPI1-MSPß interaction confirm the formation of a 1:1 complex. SPI1 and MSPαß also associate primarily as a 1:1 complex with a binding affinity similar to that of SPI1-MSPß. In addition, the SPI1-MSPαß ultracentrifuge studies reveal a low abundance 2:2 complex with ∼ 10-fold lower binding affinity compared with the 1:1 species. These results support the hypothesis that the α-chain of MSPαß mediates RON dimerization.

Systems approaches to unraveling plant metabolism: identifying biosynthetic genes of secondary metabolic pathways.

The diversity of useful compounds produced by plant secondary metabolism has stimulated broad systems biology approaches to identify the genes involved in their biosynthesis. Systems biology studies in non-model plants pose interesting but addressable challenges, and have been greatly facilitated by the ability to grow and maintain plants, develop laboratory culture systems, and profile key metabolites in order to identify critical genes involved their biosynthesis. In this chapter we describe a suite of approaches that have been useful in Actaea racemosa (L.; syn. Cimicifuga racemosa, Nutt., black coshosh), a non-model medicinal plant with no genome sequence and little horticultural information available, that have led to the development of initial gene-metabolite relationships for the production of several bioactive metabolites in this multicomponent botanical therapeutic, and that can be readily applied to a wide variety of under-characterized medicinal plants.

Calcium antagonists: a ready prescription for treating infectious diseases?

Emergence of new and medically resistant pathogenic microbes continues to escalate toward worldwide public health, wild habitat, and commercial crop and livestock catastrophes. Attempts at solving this problem with sophisticated modern biotechnologies, such as smart vaccines and microbicidal and microbistatic drugs that precisely target parasitic bacteria, fungi, and protozoa, remain promising without major clinical and industrial successes. However, discovery of a more immediate, broad spectrum prophylaxis beyond conventional epidemiological approaches might take no longer than the time required to fill a prescription at your neighborhood pharmacy. Findings from a growing body of research suggest calcium antagonists, long approved and marketed for various human cardiovascular and neurological indications, may produce safe, efficacious antimicrobial effects. As a general category of drugs, calcium antagonists include compounds that disrupt passage of Ca(2+) molecules across cell membranes and walls, sequestration and mobilization of free intracellular Ca(2+), and downstream binding proteins and sensors of Ca(2+)-dependent regulatory pathways important for proper cell function. Administration of calcium antagonists alone at current therapeutically relevant doses and schedules, or with synergistic compounds and additional antimicrobial medications, figures to enhance host immunoprotection by directly altering pathogen infection sequences, life cycles, homeostasis, antibiotic tolerances, and numerous other infective, survival, and reproductive processes. Short of being miracle drugs, calcium antagonists are welcome old drugs with new tricks capable of controlling some of the most virulent and pervasive global infectious diseases of plants, animals, and humans, including Chagas' disease, malaria, and tuberculosis.

ABRF-MIRG benchmark study: molecular interactions in a three-component system.

Protein-protein interactions identified through high-throughput proteomics efforts continue to advance our understanding of the protein interactome. In addition to highly specific protein-protein interactions, it is becoming increasingly more common for yeast two-hybrid, pull-down assays, and other proteomics techniques to identify multiple protein ligands that bind to the same target protein. A resulting challenge is to accurately characterize the assembly of these multiprotein complexes and the competition among multiple protein ligands for a given target. The Association of Biomolecular Resource Facilities-Molecular Interactions Research Group recently conducted a benchmark study to assess participants' ability to correctly describe the interactions between two protein ligands and their target protein using primarily biosensor technologies, such as surface plasmon resonance. Participants were provided with microgram quantities of three proteins (A, B, and C) and asked to determine if a ternary A-B-C complex can form or if protein-B and protein-C bind competitively to protein-A. This article will summarize the experimental approaches taken by participants to characterize the molecular interactions, the interpretation of the data, and the results obtained using different biosensor instruments.

Some new speculative ideas about the "behavioral homeostasis theory" as to how the simple learned behaviors of habituation and sensitization improve organism survival throughout phylogeny.

This paper explores further the "behavioral homeostasis theory" (BHT) regarding the evolutionary significance for organism survival of the two simple non-associative rapidly learned behaviors of habituation and sensitization. The BHT postulates that the evolutionary function of habituation and sensitization throughout phylogeny is to rapidly maximize an organism's overall readiness to cope with new stimuli and to minimize unnecessary energy expenditure. These behaviors have survived with remarkable similarity throughout phylogeny from aneural protozoa to humans. The concept of "behavioral homeostasis" emphasizes that the homeostatic process is more than just maintaining internal equilibrium in the face of changing internal and external conditions. It emphasizes the rapid internal and external effector system changes that occur to optimize organism readiness to cope with any new external stimulus situation. Truly life-threatening stimuli elicit instinctive behavior such as fight, flee, or hide. If the stimulus is not life-threatening, the organism rapidly learns to adjust to an appropriate level of overall responsiveness over stimulus repetitions. The rapid asymptotic level approached by those who decrease their overall responsiveness to the second stimulus (habituaters) and those who increase their overall responsiveness to an identical second stimulus (sensitizers) not only optimizes readiness to cope with any new stimulus situation but also reduces unnecessary energy expenditure. This paper is based on a retrospective analysis of data from 4 effector system responses to eight repetitive tone stimuli in adult human males. The effector systems include the galvanic skin response, finger pulse volume, muscle frontalis and heart rate. The new information provides the basis for further exploration of the BHT including new predictions and proposed relatively simple experiments to test them.

Structural reorganization of the interleukin-7 signaling complex.

We report here an unliganded receptor structure in the common gamma-chain (γ(c)) family of receptors and cytokines. The crystal structure of the unliganded form of the interleukin-7 alpha receptor (IL-7Rα) extracellular domain (ECD) at 2.15 Å resolution reveals a homodimer forming an "X" geometry looking down onto the cell surface with the C termini of the two chains separated by 110 Å and the dimer interface comprising residues critical for IL-7 binding. Further biophysical studies indicate a weak association of the IL-7Rα ECDs but a stronger association between the γ(c)/IL-7Rα ECDs, similar to previous studies of the full-length receptors on CD4(+) T cells. Based on these and previous results, we propose a molecular mechanism detailing the progression from the inactive IL-7Rα homodimer and IL-7Rα-γ(c) heterodimer to the active IL-7-IL-7Rα-γ(c) ternary complex whereby the two receptors undergo at least a 90° rotation away from the cell surface, moving the C termini of IL-7Rα and γ(c) from a distance of 110 Å to less than 30 Å at the cell surface. This molecular mechanism can be used to explain recently discovered IL-7- and γ(c)-independent gain-of-function mutations in IL-7Rα from B- and T-cell acute lymphoblastic leukemia patients. The mechanism may also be applicable to other γ(c) receptors that form inactive homodimers and heterodimers independent of their cytokines.

Gene identification in black cohosh (Actaea racemosa L.): expressed sequence tag profiling and genetic screening yields candidate genes for production of bioactive secondary metabolites.

Black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa, Nutt., Ranunculaceae) is a popular herb used for relieving menopausal discomforts. A variety of secondary metabolites, including triterpenoids, phenolic dimers, and serotonin derivatives have been associated with its biological activity, but the genes and metabolic pathways as well as the tissue distribution of their production in this plant are unknown. A gene discovery effort was initiated in A. racemosa by partial sequencing of cDNA libraries constructed from young leaf, rhizome, and root tissues. In total, 2,066 expressed sequence tags (ESTs) were assembled into 1,590 unique genes (unigenes). Most of the unigenes were predicted to encode primary metabolism genes, but about 70 were identified as putative secondary metabolism genes. Several of these candidates were analyzed further and full-length cDNA and genomic sequences for a putative 2,3 oxidosqualene cyclase (CAS1) and two BAHD-type acyltransferases (ACT1 and HCT1) were obtained. Homology-based PCR screening for the central gene in plant serotonin biosynthesis, tryptophan decarboxylase (TDC), identified two TDC-related sequences in A. racemosa. CAS1, ACT1, and HCT1 were expressed in most plant tissues, whereas expression of TDC genes was detected only sporadically in immature flower heads and some very young leaf tissues. The cDNA libraries described and assorted genes identified provide initial insight into gene content and diversity in black cohosh, and provide tools and resources for detailed investigations of secondary metabolite genes and enzymes in this important medicinal plant.

Structure of PqsD, a Pseudomonas quinolone signal biosynthetic enzyme, in complex with anthranilate.

Pseudomonas quinolone signal (PQS), 2-heptyl-3-hydroxy-4-quinolone, is an intercellular alkyl quinolone signaling molecule produced by the opportunistic pathogen Pseudomonas aeruginosa. Alkyl quinolone signaling is an atypical system that, in P. aeruginosa, controls the expression of numerous virulence factors. PQS is synthesized from the tryptophan pathway intermediate, anthranilate, which is derived either from the kynurenine pathway or from an alkyl quinolone specific anthranilate synthase encoded by phnAB. Anthranilate is converted to PQS by the enzymes encoded by the pqsABCDE operon and pqsH. PqsA forms an activated anthraniloyl-CoA thioester that shuttles anthranilate to the PqsD active site where it is transferred to Cys112 of PqsD. In the only biochemically characterized reaction, a condensation then occurs between anthraniloyl-PqsD and malonyl-CoA or malonyl-ACP, a second PqsD substrate, forming 2,4-dihydroxyquinoline (DHQ). The role PqsD plays in the biosynthesis of other alkyl quinolones, such as PQS, is unclear, though it has been reported to be required for their production. No evidence exists that DHQ is a PQS precursor, however. Here we present a structural and biophysical characterization of PqsD that includes several crystal structures of the enzyme, including that of the PqsD-anthranilate covalent intermediate and the inactive Cys112Ala active site mutant in complex with anthranilate. The structure reveals that PqsD is structurally similar to the FabH and chalcone synthase families of fatty acid and polyketide synthases. The crystallographic asymmetric unit contains a PqsD dimer. The PqsD monomer is composed of two nearly identical approximately 170-residue alphabetaalphabetaalpha domains. The structures show anthranilate-liganded Cys112 is positioned deep in the protein interior at the bottom of an approximately 15 A long channel while a second anthraniloyl-CoA molecule is waiting in the cleft leading to the protein surface. Cys112, His257, and Asn287 form the FabH-like catalytic triad of PqsD. The C112A mutant is inactive, although it still reversibly binds anthraniloyl-CoA. The covalent complex between anthranilate and Cys112 clearly illuminates the orientation of key elements of the PqsD catalytic machinery and represents a snapshot of a key point in the catalytic cycle.

Divergence of function in the hot dog fold enzyme superfamily: the bacterial thioesterase YciA.

Thioesters play a central role in the cells where they participate in metabolism, membrane synthesis, signal transduction, and gene regulation. Thioesters are converted to the thiol and carboxylic acid components by thioesterase-catalyzed hydrolysis. Here we examine the biochemical and biological function of the hot dog fold thioesterase YciA (EcYciA) from Escherichia coli and its close sequence homologue HI0827 from Haemophilus influenzae (HiYciA). The quaternary structure of HiYciA was determined, using equilibrium sedimentation techniques, to be a homohexamer. Mass spectral and (31)P NMR analysis of purified HiYciA revealed a bound CoA ligand. Kinetic analyses showed that CoA is a strong feedback inhibitor. YciA thioesterase activity toward acyl-CoA substrates was determined using steady-state kinetic methods. The k cat and k cat/ K m values obtained reveal a striking combination of high catalytic efficiency and low substrate specificity. The substrate activity of propionyl-s- N-acetylcysteine was found to be negligible and that of n-butyryl-pantetheinephosphate low, and therefore, it is evident YciA does not target acylated ACPs or other acylated proteins as substrates. The results from bioinformatic analysis of the biological distribution and genome contexts of yciAs are reported. We conclude that YciA is responsible for the efficient, "seemingly" indiscriminant, CoA-regulated hydrolysis of cellular acyl-CoA thioesters in a wide range of bacteria and hypothesize that this activity may support membrane biogenesis.

NMR structure of HI0004, a putative essential gene product from Haemophilus influenzae, and comparison with the X-ray structure of an Aquifex aeolicus homolog.

The solution structure of the 154-residue conserved hypothetical protein HI0004 has been determined using multidimensional heteronuclear NMR spectroscopy. HI0004 has sequence homologs in many organisms ranging from bacteria to humans and is believed to be essential in Haemophilus influenzae, although an exact function has yet to be defined. It has a alpha-beta-alpha sandwich architecture consisting of a central four-stranded beta-sheet with the alpha2-helix packed against one side of the beta-sheet and four alpha-helices (alpha1, alpha3, alpha4, alpha5) on the other side. There is structural homology with the eukaryotic matrix metalloproteases (MMPs), but little sequence similarity except for a conserved region containing three histidines that appears in both the MMPs and throughout the HI0004 family of proteins. The solution structure of HI0004 is compared with the X-ray structure of an Aquifex aeolicus homolog, AQ_1354, which has 36% sequence identity over 148 residues. Despite this level of sequence homology, significant differences exist between the two structures. These differences are described along with possible functional implications of the structures.

Structure of the phenazine biosynthesis enzyme PhzG.

PhzG is a flavin-dependent oxidase that is believed to play a role in phenazine antibiotic synthesis in various bacteria, including Pseudomonas. Phenazines are chorismic acid derivatives that provide the producing organisms, including the opportunistic pathogen P. aeruginosa, with a competitive growth advantage. Here, the crystal structures of PhzG from both P. aeruginosa and P. fluorescens solved in an unliganded state at 1.9 and 1.8 A resolution, respectively, are described. Although the specific reaction in phenazine biosynthesis catalyzed by PhzG is unknown, the structural data indicates that PhzG is closely related to pyridoxine-5'-phosphate oxidase, the Escherichia coli pdxH gene product, which catalyzes the final step in pyridoxal-5'-phosphate (PLP) biosynthesis. A previous proposal suggested that the physiological substrate of PhzG to be 2,3-dihydro-3-hydroxyanthranilic acid (DHHA), a phenazine precursor produced by the sequential actions of the PhzE and PhzD enzymes on chorismate, and that two DHHA molecules dimerized in another enzyme-catalyzed reaction to yield phenazine-1-carboxylate. However, it was not possible to demonstrate any in vitro activity upon incubation of PhzG and DHHA. Interestingly, analysis of the in vitro activities of PhzG in combination with PhzF suggests that PhzF acts on DHHA and that PhzG then reacts with a non-aromatic tricyclic phenazine precusor to catalyze an oxidation/aromatization reaction that yields phenazine-1-carboxylate. It is proposed that phzG arose by duplication of pdxH and that the subtle differences seen between the structures of PhzG and PdxH correlate with the loss of the ability of PhzG to catalyze PLP formation. Sequence alignments and superimpositions of the active sites of PhzG and PdxH reveal that the residues that form a positively charged pocket around the phosphate of PLP in the PdxH-PLP complex are not conserved in PhzG, consistent with the inability of phosphorylated compounds to serve as substrates for PhzG.

Structure and function of the phenazine biosynthesis protein PhzF from Pseudomonas fluorescens 2-79.

Phenazines, including pyocyanin and iodonin, are biologically active compounds that are believed to confer producing organisms with a competitive growth advantage, and also are thought to be virulence factors in certain diseases including cystic fibrosis. The basic, tricyclic phenazine ring system is synthesized in a series of poorly characterized steps by enzymes encoded in a seven-gene cistron in Pseudomonas and other organisms. Despite the biological importance of these compounds, and our understanding of their mode of action, the biochemistry and mechanisms of phenazine biosynthesis are not well resolved. Here we report the 1.8 A crystal structure of PhzF, a key enzyme in phenazine biosynthesis, solved by molecular replacement. PhzF is structurally similar to the lysine biosynthetic enzyme diaminopimelate epimerase, sharing an unusual fold consisting of two nearly identical domains with the active site located in an occluded cleft between the domains. Unlike diaminopimelate epimerase, PhzF is a dimer in solution. The two apparently independent active sites open toward opposite sides of the dimer and are occupied by sulfate ions in the structure. In vitro experiments using a mixture of purified PhzF, -A, -B, and -G confirm that phenazine-1-carboxylic acid (PCA) is readily produced from trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) without aid of other cellular factors. PhzA, -B, and -G have no activity toward DHHA. However, in the presence of PhzF, individually or in combinations, they accelerate the formation of PCA from DHHA and therefore appear to function after the action of PhzF. Surprisingly, PhzF is itself capable of producing PCA, albeit slowly, from DHHA. These observations suggest that PhzF catalyzes the initial step in the conversion of DHHA to PCA, probably via a rearrangement reaction yielding the more reactive 3-oxo analogue of DHHA, and that subsequent steps can occur spontaneously. A hypothetical model for how DHHA binds to the PhzF active site suggests that Glu45 and Asp208 could act as general acid-base catalysts in a rearrangement reaction. Given that four reactions lie between DHHA and PCA, ketone formation, ring formation, decarboxylation, and oxidation, we hypothesize that the similar PhzA and -B proteins catalyze ring formation and thus may be more than noncatalytic accessory proteins. PhzG is almost certainly an oxidase and is predicted to catalyze the final oxidation/aromatization reaction.