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Identification of Residues of the Kid Toxin Involved in Autoregulation of the parD System. Marc Lemonnier, 2004.The toxin-antitoxin system parD (kis kid) of plasmid R1 is coregulated by the coordinated action of its two gene products . Here we describe the isolation and the in vivo characterization of three single-amino-acid changes in the Kid toxin, G4E, C74Y, and E91K, that affect the coregulatory activity but preserve the toxicity of the protein . Purification, Characterization, and Sequencing of an Extracellular Cold-Active Aminopeptidase Produced by Marine Psychrophile Colwellia psychrerythraea Strain 34H. Adrienne L. Huston, 2004.The limited database on cold-active extracellular proteases from marine bacteria was expanded by successful purification and initial biochemical and structural characterization of a family M1 aminopeptidase (designated ColAP) produced by the marine psychrophile Colwellia psychrerythraea strain 34H . The 71-kDa enzyme displayed a low optimum temperature (19°C) and narrow pH range (pH 6 to 8.5) for activity and greater thermolability than other extracellular proteases . Sequencing of the gene encoding ColAP revealed a predicted amino acid sequence with the highest levels of identity (45 to 55%) to M1 aminopeptidases from mesophilic members of the  subclass of the Proteobacteria and the next highest levels of identity (35 to 36%) to leukotriene A4 hydrolases from mammalian sources . Compared to mesophilic homologs, ColAP had structural differences thought to increase the flexibility for activity in the cold; for example, it had fewer proline residues, fewer ion pairs, and a lower hydrophobic residue content . In addition to intrinsic properties that determine enzyme activity and stability, we also investigated effects of extracellular polymeric substances (EPS) from spent culture medium of strain 34H on ColAP activity at an environmentally relevant temperature (0°C) and at 45°C (the maximum temperature for activity) . In both cases, ColAP stability increased significantly in the presence of EPS, indicating the importance of considering environmentally relevant extrinsic factors when enzyme structure and function are investigated .
The Extracellular Transport Signal of the Vibrio cholerae Endochitinase (ChiA) Is a Structural Motif Located between Amino Acids 75 and 555. Jason P. Folster, 2002.ChiA, an 88-kDa endochitinase encoded by the chiA gene of the gram-negative enteropathogen Vibrio cholerae, is secreted via the eps-encoded main terminal branch of the general secretory pathway (GSP), a mechanism which also transports cholera toxin . To localize the extracellular transport signal of ChiA that initiates transport of the protein through the GSP, a chimera comprised of ChiA fused at the N terminus with the maltose-binding protein (MalE) of Escherichia coli and fused at the C terminus with a 13-amino-acid epitope tag (E-tag) was expressed in strain 569B(chiA::Kanr), a chiA-deficient but secretion-competent mutant of V . cholerae . Fractionation studies revealed that blockage of the natural N terminus and C terminus of ChiA did not prevent secretion of the MalE-ChiA-E-tag chimera . To locate the amino acid sequences which encoded the transport signal, a series of truncations of ChiA were engineered . Secretion of the mutant polypeptides was curtailed only when ChiA was deleted from the N terminus beyond amino acid position 75 or from the C terminus beyond amino acid 555 . A mutant ChiA comprised of only those amino acids was secreted by wild-type V . cholerae but not by an epsD mutant, establishing that amino acids 75 to 555 independently harbored sufficient structural information to promote secretion by the GSP of V . cholerae . Cys77 and Cys537, two cysteines located just within the termini of ChiA(75-555), were not required for secretion, indicating that those residues were not essential for maintaining the functional activity of the ChiA extracellular transport signal .
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