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Novel Polyketide Synthase from Nectria haematococca. Stephane Graziani, 2004.We identified a polyketide synthase (PKS) gene, pksN, from a strain of Nectria haematococca by complementing a mutant unable to synthesize a red perithecial pigment . pksN encodes a 2,106-amino-acid polypeptide with conserved motifs characteristic of type I PKS enzymatic domains: ß-ketoacyl synthase, acyltransferase, duplicated acyl carrier proteins, and thioesterase . The pksN product groups with the Aspergillus nidulans WA-type PKSs involved in conidial pigmentation and melanin, bikaverin, and aflatoxin biosynthetic pathways . Inactivation of pksN did not cause any visible change in fungal growth, asexual sporulation, or ascospore formation, suggesting that it is involved in a specific developmental function . We propose that pksN encodes a novel PKS required for the perithecial red pigment biosynthesis . Directed Evolution of Toluene ortho-Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation. Keith A. Canada, 2002.Trichloroethylene (TCE) is the most frequently detected groundwater contaminant, and 1-naphthol is an important chemical manufacturing intermediate . Directed evolution was used to increase the activity of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 for both chlorinated ethenes and naphthalene oxidation . When expressed in Escherichia coli, the variant TOM-Green degraded TCE (2.5 ± 0.3 versus 1.39 ± 0.05 nmol/min/mg of protein), 1,1-dichloroethylene, and trans-dichloroethylene more rapidly . Whole cells expressing TOM-Green synthesized 1-naphthol at a rate that was six times faster than that mediated by the wild-type enzyme at a concentration of 0.1 mM (0.19 ± 0.03 versus 0.029 ± 0.004 nmol/min/mg of protein), whereas at 5 mM, the mutant enzyme was active (0.07 ± 0.03 nmol/min/mg of protein) in contrast to the wild-type enzyme, which had no detectable activity . The regiospecificity of TOM-Green was unchanged, with greater than 97% 1-naphthol formed . The beneficial mutation of TOM-Green is the substitution of valine to alanine in position 106 of the  -subunit of the hydroxylase, which appears to act as a smaller "gate" to the diiron active center . This hypothesis was supported by the ability of E . coli expressing TOM-Green to oxidize the three-ring compounds, phenanthrene, fluorene, and anthracene faster than the wild-type enzyme . These results show clearly that random, in vitro protein engineering can be used to improve a large multisubunit protein for multiple functions, including environmental restoration and green chemistry .
Salmonella enterica Serovar Typhimurium rdoA Is Growth Phase Regulated and Involved in Relaying Cpx-Induced Signals. P. Suntharalingam, 2003.The disulfide oxidoreductase, DsbA, mediates disulfide bond formation in proteins as they enter or pass through the periplasm of gram-negative bacteria . Although DsbA function has been well characterized, less is known about the factors that control its expression . Previous studies with Escherichia coli demonstrated that dsbA is part of a two-gene operon that includes an uncharacterized, upstream gene, yihE, that is positively regulated via the Cpx stress response pathway . To clarify the role of the yihE homologue on dsbA expression in Salmonella enterica serovar Typhimurium, the effect of this gene (termed rdoA) on the regulation of dsbA expression was investigated . Transcriptional assays assessing rdoA promoter activity showed growth phase-dependent expression with maximal activity in stationary phase . Significant quantities of rdoA and dsbA transcripts exist in serovar Typhimurium, but only extremely low levels of rdoA-dsbA cotranscript were detected . Activation of the Cpx system in serovar Typhimurium increased synthesis of both rdoA- and dsbA-specific transcripts but did not significantly alter the levels of detectable cotranscript . These results indicate that Cpx-mediated induction of dsbA transcription in serovar Typhimurium does not occur through an rdoA-dsbA cotranscript . A deletion of the rdoA coding region was constructed to definitively test the relevance of the rdoA-dsbA cotranscript to dsbA expression . The absence of RdoA affects DsbA expression levels when the Cpx system is activated, and providing rdoA in trans complements this phenotype, supporting the hypothesis that a bicistronic mechanism is not involved in serovar Typhimurium dsbA regulation . The rdoA null strain was also shown to be altered in flagellar phase variation . First it was found that induction of the Cpx stress response pathway switched flagellar synthesis to primarily phase 2 flagellin, and this effect was then found to be abrogated in the rdoA null strain, suggesting the involvement of RdoA in mediating Cpx-related signaling .
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