Structural and biochemical characterization of citrate binding to AtPPC3, a plant-type phosphoenolpyruvate carboxylase from Arabidopsis thaliana.

Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated cytosolic enzyme situated at a crucial branch point of central plant metabolism. The structure of AtPPC3, a C3 PEPC isozyme of the model plant Arabidopsis thaliana, in complex with the inhibitors aspartate and citrate was solved at 2.2-Å resolution. This represents the first PEPC structure with citrate bound. Aspartate and citrate binding sites are in close proximity (5.1-5.3 Å) and interactions between citrate and specific residues were identified. Citrate functions as a mixed (allosteric) inhibitor as it reduced AtPPC3's Vmax while increasing Km(PEP) values. The PEP saturation data gave an excellent fit to the mixed inhibition model, yielding Ki and Ki' (citrate) values of 9.3 and 42.5 mM, respectively. Citrate and aspartate inhibition of AtPPC3 was non-additive, likely due to their closely positioned binding sites, their similar negative charge, and type of binding residues. Fewer interactions and lower affinity for citrate support its observed weaker inhibition of AtPPC3 relative to aspartate. Citrate does not appear to induce further conformational change beyond aspartate owing to the similar structural mechanism of inhibition. AtPPC3 largely exhibits root-specific expression in Arabidopsis, where it is markedly upregulated during stresses such as excessive salinity or nutritional Pi deprivation that necessitate large increases in anaplerotic PEP carboxylation. The cytosolic citrate concentration of potato tubers suggests that AtPPC3's inhibition by citrate may be physiologically relevant. Our results provide novel insights into the structural basis of allosteric PEPC control and the kinetic effects brought about upon inhibitor binding.

Function Coupling Mechanism of PhuS and HemO in Heme Degradation.

Most bacteria possess only one heme-degrading enzyme for obtaining iron, however few bacteria such as Pseudomonas aeruginosa express two, namely PhuS and HemO. While HemO is a well-known heme oxygenase, previously we discovered that PhuS also possesses heme degradation activity and generates verdoheme, an intermediate of heme breakdown. To understand the coexistence of these two enzymes, using the DFT calculation we reveal that PhuS effectively enhances heme degradation through its participation in heme hydroxylation, the rate limiting reaction. Heme is converted to verdoheme in this reaction and the energy barrier for PhuS is substantially lower than for HemO. Thus, HemO is mainly involved in the ring opening reaction which converts verdoheme to biliverdin and free iron. Our kinetics experiments show that, in the presence of both PhuS and HemO, complete degradation of heme to biliverdin is enhanced. We further show that PhuS is more active than HemO using heme as a substrate and generates more CO. Combined experimental and theoretical results directly identify function coupling of this two-enzyme system, resulting in more efficient heme breakdown and utilization.

Growth of Diffraction-Quality Protein Crystals Using a Harvestable Microfluidic Device.

Protein crystallization is the major bottleneck in the entire process of protein crystallography, and obtaining diffraction-quality crystals can be unpredictable and sometimes exceptionally difficult, requiring many rounds of high-throughput screening. Recently, a more time- and cost-saving strategy to use the commercially available microfluidic devices called Crystal Formers has emerged. Herein we show the application of such a device using a protein from Legionella pneumophila called LidL that is predicted to be involved in the ability to efficiently manipulate host cell trafficking events once internalized by the host cell. After setting up just one 96-channel Crystal Former tray, we were able to obtain a diffraction-quality crystal that diffracted to 2.76 Å. These results show that Crystal Formers can be used to screen and optimize crystals to directly produce crystals for structure determination.

Structural analysis and identification of PhuS as a heme-degrading enzyme from Pseudomonas aeruginosa.

Bacterial pathogens require iron for proliferation and pathogenesis. Pseudomonas aeruginosa is a prevalent Gram-negative opportunistic human pathogen that takes advantage of immunocompromised hosts and encodes a number of proteins for uptake and utilization of iron. Here we report the crystal structures of PhuS, previously known as the cytoplasmic heme-trafficking protein from P. aeruginosa, in both the apo- and the holo-forms. In comparison to its homologue ChuS from Escherichia coli O157:H7, the heme orientation is rotated 180° across the α-γ axis, which may account for some of the unique functional properties of PhuS. In contrast to previous findings, heme binding does not result in an overall conformational change of PhuS. We employed spectroscopic analysis and CO measurement by gas chromatography to analyze heme degradation, demonstrating that PhuS is capable of degrading heme using ascorbic acid or cytochrome P450 reductase-NADPH as an electron donor and produces five times more CO than ChuS. Addition of catalase slows down but does not stop PhuS-catalyzed heme degradation. Through spectroscopic and mass spectrometry analysis, we identified the enzymatic product of heme degradation to be verdoheme. These data taken together suggest that PhuS is a potent heme-degrading enzyme, in addition to its proposed heme-trafficking function.