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A mitogen-activated protein kinase (MAPK), Pfmap2, has been identified in Plasmodium falciparum. However, its bona fide activator remains elusive as no MAPK kinase (MAPKK) homologues have been found so far. Instead, Pfnek3, a NIMA (never in mitosis, Aspergillus)-related kinase, was earlier reported to display a MAPKK-like activity due to its activating effect on Pfmap2. In this study, the regulatory mechanism of Pfnek3 was investigated. Pfnek3 was found to possess a SSEQSS motif within its activation loop that fulfills the consensus SXXXS/T phospho-activating sequence of MAPKKs. Functional analyses of the SSEQSS motif by site-directed mutagenesis revealed that phosphorylation of residues S221 and S226 is essential for mediating Pfnek3 activity. Moreover, via tandem mass-spectrometry, residue T82 was uncovered as an additional phosphorylation site involved in Pfnek3 activation. Collectively, these results provide valuable insights into the potential in vivo regulation of Pfnek3, with residues T82, S221 and S226 functioning as phospho-activating sites.

Streptomyces clavuligerus deacetoxycephalosporin C synthase (ScDAOCS) is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid, which is a precursor for cephalosporin synthesis. Single mutations of six amino acid residues, V275, C281, N304, I305, R306, and R307, were previously shown to result in enhanced levels of ampicillin conversion, with activities ranging from 129 to 346% of the wild-type activity. In this study, these mutations were paired to investigate their effects on enzyme catalysis. The bioassay results showed that the C-terminal mutations (N304X [where X is alanine, leucine, methionine, lysine, or arginine], I305M, R306L, and R307L) in combination with C281Y substantially increased the conversion of ampicillin; the activity was up to 491% of the wild-type activity. Similar improvements were observed for converting carbenicillin (up to 1,347% of the wild-type activity) and phenethicillin (up to 1,109% of the wild-type activity). Interestingly, the N304X R306L double mutants exhibited lower activities for penicillin G conversion, and activities that were 40 to 114% of wild-type enzyme activity were detected. Based on kinetic studies using ampicillin, it was clear that the increases in the activities of the double mutants relative to those of the corresponding single mutants were due to enhanced substrate binding affinities. These results also validated the finding that the N304R and I305M mutations are ideal for increasing the substrate binding affinity and turnover rate of the enzyme, respectively. This study provided further insight into the structurefunction interaction of ScDAOCS with different penicillin substrates, thus providing a useful platform for further rational modification of its enzymatic properties.

There is urgent need for effective bactericidal agents for use in real commercial formulations since many old disinfectants, such as halogenated compounds, are now banned. Cationic polymers may have good bactericidal properties in pure water or buffer, but typically become ineffective in the presence of anionic surfactants that are widely used in many commercial formulations. Here, we discover that polyion complex (PIC) nanoparticles formed by cationic polymers of Poly(3-AcrylaMidoPropyl) TriMethylAmmonium chloride (PAMPTMA) in the presence of anionic surfactants display promising fast-bactericidal effect (> 99.99 % killing within 10-min treatment) on Gram-negative Escherichia coli (E. coli, ATCC ® 8739™). To examine the influence of hydrophobicity on bactericidal property, we synthesize PAMPTMA-b-Poly(Butyl MethAcrylate) and discover that increasing hydrophobicity has little influence on bactericidal property of PIC nanoparticles. Mechanism study shows that cationic PIC nanoparticles rapidly cause significant pores both in the outer-and inner-membranes because of their large size and high local concentration of positive charges. The rapid membrane pore formation results in fast cell death. The discovery --certain cationic polymers when formulated with anionic surfactants are even more bactericidal than neat cationic polymers alone --paves the way for potential applications of synthetic cationic polymers in commercial formulations.