The canonical mitogen-activated protein kinase (MAPK) signal cascade was previously suggested to be atypical in the malaria parasite. This raises queries on the existence of alternative mediators of plasmodial MAPK pathways. This study describes, Pfnek3, a malarial protein kinase belonging to the NIMA (Never in Mitosis, Aspergillus) family. Endogenous Pfnek3 is expressed during late asexual to gametocyte stages and lacks some classical protein kinase sequence motifs. Moreover, Pfnek3 is phylogenetically distant from mammalian NIMA-kinases. Recombinant Pfnek3 was able to phosphorylate and stimulate a malarial MAPK (Pfmap2). Contrastingly, this was not observed with two other kinases, Pfmap1 and human MAPK1, suggesting that the Pfnek3-Pfmap2 interaction may be specific for Pfmap2 regulation. In summary, our data reveal a malarial NIMA-kinase with the potential to regulate a MAPK. Possessing biochemical properties divergent from classical mammalian NIMA-kinases, Pfnek3 could potentially be an attractive target for parasite-selective anti-malarials.

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~~Malate synthase is a key enzyme of the glyoxylate cycle, which is an anaplerotic pathway essential for growth on acetate as the sole carbon source. The aceB gene, encoding malate synthase from Streptomyces clavuligems NRRL 3585, was cloned using PCR and fully sequenced. The ORF obtained encodes 541 amino acids with a deduced M, of 6OOOO, consistent with the' observed M, (62000-64000) of most malate synthase enzymes reported so far. The aceB gene has a high G+C content (71.5 molO/O), especially in the third codon position. A 50 bp region upstream of the malate synthase ORF was predicted to be a prokaryotic promoter region. The relationship between carbon source, antibiotic (cephalosporin) biosynthesis and malate synthase activity was investigated. Growth of S. clavuligerus on acetate as the major carbon source was delayed, compared to that on glycerol. Furthermore, high levels of malate synthase activity were associated with the presence of acetate in the growth medium. Growth on acetate also resulted in lower levels of cephalosporin production, compared to that on glycerol. The cloned 5. clavuligerus aceB gene was expressed in Escherichia coli BL2l (DE3). Transformants exhibited an approximately 713old increase in malate synthase activity, compared to the control, thereby demonstrating high-level expression of soluble and enzymically active malate synthase in the heterologous host.

Industrially, the use of high temperatures (40-60 degrees C) in the L: -malate production process could result in rapid inactivation of the mesophilic fumarases, warranting constant replenishment of the biocatalyst. Thus, a thermostable fumarase C that is active and stable at high temperatures would be ideal. Biochemical studies using recombinant fumarase C from thermophilic Streptomyces thermovulgaris (stFUMC) indicated that it was optimally active at 50 degrees C and highly stable even after 24 h of incubation at 40 degrees C. The same gene from mesophilic Streptomyces coelicolor (scfumC) was also cloned and expressed as soluble proteins for comparison in thermal properties of both enzymes. In contrast to stFUMC, scFUMC exhibited a lower temperature optima of 30 degrees C and was rapidly denatured at 50 degrees C. The specific activity of stFUMC was also higher than that of scFUMC by 20-fold. After primary sequence comparison, three hydrophilic amino acid residues, R163, E170 and S347, were forged into the thermolabile scFUMC either singly or in combination for the investigation of their contributions in the thermal properties of the mutant enzymes. Of the mutants studied, the A347S scFUMC mutant resulted in the highest increase in optimum temperature of 10 degrees C and a fourfold enhancement in specific activity. G163R/G170E and G163R/G170E/A347S scFUMC mutants are more thermostable than wild-type scFUMC. These findings support stFUMC as a highly efficient, thermostable fumarase C with industrial potential and suggest that R163, E170 and S347 are involved in the enhancement of thermal properties in fumarase C.