title: Purification, characterization, and sequencing of an extracellular cold-active aminopeptidase produced by marine psychrophile Colwellia psychrerythraea strain 34H.
abstract: 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 degrees 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 gamma subclass of the Proteobacteria and the next highest levels of identity (35 to 36%) to leukotriene A(4) 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 degrees C) and at 45 degrees 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.
authors.affiliation: University of Washington School of Oceanography, Seattle, Washington 98195,USA. ahuston@u.washington.edu
authors.forename: Adrienne L
authors.initials: AL
authors.lastname: Huston
authors.forename: Barbara
authors.initials: B
authors.lastname: Methe
authors.forename: Jody W
authors.initials: JW
authors.lastname: Deming
mesh_terms: D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003080:Cold Temperature; D003470:Culture Media; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000254:growth & development; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D012623:Seawater / Q000382:microbiology*; D017422:Sequence Analysis, DNA
chemical_list: D003470:Culture Media; D000626:Aminopeptidases