Structural and molecular dynamic studies of Pseudomonas aeruginosa OdaA reveal the regulation role of a C-terminal hinge element.

BACKGROUND: Crotonase superfamily members exhibit great catalytic diversity towards various acyl-CoA substrates. A common CoA moiety binding pattern is usually observed in this family, understanding the substrate-binding mechanism would facilitate the rational engineering of crotonases for improved properties. METHODS: We applied X-ray crystallography to investigate a putative enoyl-CoA hydratase/isomerase OdaA in Pseudomonas aeruginosa. Thermal shift assay (TSA) were performed to explore the binding of OdaA with CoA thioester substrates. Furthermore, we performed molecular dynamics (MD) simulations to elucidate the dynamics of its CoA-binding site. RESULTS: We solved the crystal structures of the apo and CoA-bound OdaA. Thermal shift assay (TSA) showed that CoA thioester substrates bind to OdaA with a different degree. MD simulations demonstrated that the C-terminal alpha helix underwent a structural transition and a hinge region would associate with this conformational change. CONCLUSIONS: TSA in combination with MD simulations elucidate that the dynamics of C-terminal alpha helix in CoA-binding, and a hinge region play an important role in conformational change. GENERAL SIGNIFICANCE: Those results help to extend our knowledge about the nature of crotonases and would be informative for future mechanistic studies and industry applications.

Chaperone-tip adhesin complex is vital for synergistic activation of CFA/I fimbriae biogenesis.

Colonization factor CFA/I defines the major adhesive fimbriae of enterotoxigenic Escherichia coli and mediates bacterial attachment to host intestinal epithelial cells. The CFA/I fimbria consists of a tip-localized minor adhesive subunit, CfaE, and thousands of copies of the major subunit CfaB polymerized into an ordered helical rod. Biosynthesis of CFA/I fimbriae requires the assistance of the periplasmic chaperone CfaA and outer membrane usher CfaC. Although the CfaE subunit is proposed to initiate the assembly of CFA/I fimbriae, how it performs this function remains elusive. Here, we report the establishment of an in vitro assay for CFA/I fimbria assembly and show that stabilized CfaA-CfaB and CfaA-CfaE binary complexes together with CfaC are sufficient to drive fimbria formation. The presence of both CfaA-CfaE and CfaC accelerates fimbria formation, while the absence of either component leads to linearized CfaB polymers in vitro. We further report the crystal structure of the stabilized CfaA-CfaE complex, revealing features unique for biogenesis of Class 5 fimbriae.

Structural and biochemical analysis of 1-Cys peroxiredoxin ScPrx1 from Saccharomyces cerevisiae mitochondria.

BACKGROUND: ScPrx1 is a yeast mitochondrial 1-Cys peroxiredoxins (Prx), a type of Prx enzyme which require thiol-containing reducing agents to resolve its peroxidatic cysteine. ScPrx1 plays important role in protection against oxidative stress. Mitochondrial thioredoxin ScTrx3 and glutathione have been reported to be the physiological electron donor for ScPrx1. However, the mechanism underlying their actions, especially the substrate recognition of ScPrx1 requires additional elucidation. METHODS: The structure of ScPrx1 was obtained through crystallization experiments. The oligomeric state of ScPrx1 was monitored by Blue-Native PAGE. Mutations were generated by the QuikChange PCR-based method. The ScPrx1 activity assay was carried out by measuring the change of 340 nm absorption of the NADPH oxidation. RESULTS: ScPrx1 exist as a homodimer in solution. The structure adopts a typical Prx-fold core which is preceded by an N-terminal ß-hairpin and has a C-terminal extension. Mutations (Glu94Ala, Arg198Ala and Trp126) close to the active site could enhance the catalytic efficiency of ScPrx1 while His83Ala and mutations on α4-ß6 region exhibited reduced activity. The biochemical data also show that the deletion or mutations on ScPrx1 C-terminal have 2-4.56 fold increased activity. CONCLUSION: We inferred that conformational changes of ScPrx1 C-terminal segment were important for its reaction, and the α4-ß6 loop regions around the ScPrx1 active sites were important for the catalytic function of ScPrx1. Collectively, these structural features provides a basis for understanding the diverse reductant species usage in different 1-Cys Prxs.

Structural and Functional Insights into PpgL, a Metal-Independent ß-Propeller Gluconolactonase That Contributes to Pseudomonas aeruginosa Virulence.

Biofilm formation is a critical determinant in the pathopoiesis of Pseudomonas aeruginosa It could significantly increase bacterial resistance to drugs and host defense. Thus, inhibition of biofilm matrix production could be regarded as a promising attempt to prevent colonization of P. aeruginosa and the subsequent infection. PpgL, a periplasmic gluconolactonase, has been reported to be involved in P. aeruginosa quorum-sensing (QS) system regulation. However, the detailed function and catalysis mechanism remain elusive. Here, the crystal structure of PpgL is described in the current study, along with biochemical analysis, revealing that PpgL is a typical ß-propeller enzyme with unique metal-independent lactone hydrolysis activity. Consequently, comparative analysis of seven-bladed propeller lactone-catalyzing enzymes and mutagenesis studies identify the critical sites which contribute to the diverse catalytic and substrate recognition functions. In addition, the reduced biofilm formation and attenuated invasion phenotype resulting from deletion of ppgL confirm the importance of PpgL in P. aeruginosa pathogenesis. These results suggest that PpgL is a potential target for developing new agents against the diseases caused by P. aeruginosa.

Structural characterization of a Δ3, Δ2-enoyl-CoA isomerase from Pseudomonas aeruginosa: implications for its involvement in unsaturated fatty acid metabolism.

Gene PA4980 from Pseudomonas aeruginosa encodes a putative enoyl-coenzyme A hydratase/isomerase that is associated with the function of the biofilm dispersion-inducing signal molecule cis-2-decenoic acid. To elucidate the role of PA4980 in cis-2-decenoic acid biosynthesis, we reported the crystal structure of its protein product at 2.39 Å. The structural analysis and substrate binding prediction suggest that it acts as a monofunctional enoyl-coenzyme A isomerase, implicating an alternative pathway of the cis-2-decenoic acid synthesis.

Author Correction: Structural and functional studies on Pseudomonas aeruginosa DspI: implications for its role in DSF biosynthesis.

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Structural and functional studies on Pseudomonas aeruginosa DspI: implications for its role in DSF biosynthesis.

DspI, a putative enoyl-coenzyme A (CoA) hydratase/isomerase, was proposed to be involved in the synthesis of cis-2-decenoic acid (CDA), a quorum sensing (QS) signal molecule in the pathogen Pseudomonas aeruginosa (P. aeruginosa). The present study provided a structural basis for the dehydration reaction mechanism of DspI during CDA synthesis. Structural analysis reveals that Glu126, Glu146, Cys127, Cys131 and Cys154 are important for its enzymatic function. Moreover, we show that the deletion of dspI results in a remarkable decreased in the pyoverdine production, flagella-dependent swarming motility, and biofilm dispersion as well as attenuated virulence in P. aeruginosa PA14. This study thus unravels the mechanism of DspI in diffusible signal factor (DSF) CDA biosynthesis, providing vital information for developing inhibitors that interfere with DSF associated pathogenicity in P. aeruginosa.

Mechanistic insights into the allosteric regulation of Pseudomonas aeruginosa aspartate kinase.

In plants and microorganisms, aspartate kinase (AK) catalyzes an initial commitment step of the aspartate family amino acid biosynthesis. Owing to various structural organizations, AKs from different species show tremendous diversity and complex allosteric controls. We report the crystal structure of AK from Pseudomonas aeruginosa (PaAK), a typical α2ß2 hetero-tetrameric enzyme, in complex with inhibitory effectors. Distinctive features of PaAK are revealed by structural and biochemical analyses. Essentially, the open conformation of Lys-/Thr-bound PaAK structure clarifies the inhibitory mechanism of α2ß2-type AK. Moreover, the various inhibitory effectors of PaAK have been identified and a general amino acid effector motif of AK family is described.

Structure-Function Relationship of Aminopeptidase P from Pseudomonas aeruginosa.

PepP is a virulence-associated gene in Pseudomonas aeruginosa, making it an attractive target for anti-P. aeruginosa drug development. The encoded protein, aminopeptidases P (Pa-PepP), is a type of X-prolyl peptidase that possesses diverse biological functions. The crystal structure verified its canonical pita-bread fold and functional tetrameric assembly, and the functional studies measured the influences of different metal ions on the activity. A trimetal manganese cluster was observed at the active site, elucidating the mechanism of inhibition by metal ions. Additionally, a loop extending from the active site appeared to be important for specific large-substrate binding. Based on the structural comparison and bacterial invasion assays, we showed that this non-conserved surface loop was critical for P. aeruginosa virulence. Taken together, these findings can extend our understanding of the catalytic mechanism and virulence-related functions of Pa-PepP and provide a solid foundation for the design of specific inhibitors against pathogenic-bacterial infections.

[The application of perioperative clinical pathway for severe preeclampsia patients in intensive care unit].

OBJECTIVE: To evaluate the effect of implementation of perioperative clinical pathway (CP) for severe preeclampsia patients in intensive care unit (ICU), and to discuss variation factors in order to improve clinical quality. METHODS: Thirty-six patients treated in ICU in the Second Clinical Hospital of Fujian Medical University were divided into two groups according to time of 1 year before implementation of CP (from January to December in 2009, n=14) and 1 year after implementation of CP (from January to December in 2010, n=22). The length of stay in ICU, cost of hospitalization, occurrence of major complications and mortality, as well as the total effective rate of control of blood pressure in the first 3 days after operation were compared. RESULTS: Compared with the group of patients of 1 year before implementation of CP, in the group of patients of 1 year after implementation of CP, the length of stay in ICU (hours) was significantly shorter (65.5±24.9 vs. 86.3±28.2, t=2.321, P<0.05), the cost of hospitalization (yuan) was significantly lower (6 463.6±1 838.2 vs. 8 136.5±2 142.8, t=2.496, P<0.05), the occurrence rate of major complications was lower (36.4% vs. 42.8%, χ2=0.100, P>0.05), the total effective control rate of blood pressure was significant improved on the 1st and the 2nd postoperative day (1 day: 59.1% vs. 14.3%, 2 days: 86.4% vs. 50.0%, both P<0.05), but there was no significant change on the 3rd postoperative day (95.4% vs. 85.7%, P>0.05). One patient died before the application of CP, and none after its application. CONCLUSION: These results suggested that it was beneficial to implement the program in preeclampsia patients to improve medical quality.