Journal
CELLULAR AND MOLECULAR LIFE SCIENCES
Volume 68, Issue 17, Pages 2845-2857Publisher
SPRINGER BASEL AG
DOI: 10.1007/s00018-011-0724-4
Keywords
Protein engineering; Site-directed mutagenesis; Circular permutation; Limited proteolysis; Circular dichroism; Biological activity; Antimicrobial proteins; Enterocin; Circular protein
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Funding
- Ministerio de Ciencia e Innovacion, Spain [BIO2005-01544, BIO2008-01708]
- Junta de Andalucia [PAI CVI 160]
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Bacteriocin AS-48 is an intriguing molecule because of its unique structural characteristics, genetic regulation, broad activity spectrum, and potential biotechnological applications. It was the first reported circular bacteriocin and has been undoubtedly the best characterized for the last 25 years. Thus, AS-48 is the prototype of circular bacteriocins (class IV), for which the structure and genetic regulation have been elucidated. This review discusses the state-of-the-art in genetic engineering with regard to this circular protein, with the use of site-directed mutagenesis and circular permutation. Mutagenesis studies have been used to unravel the role of (a) different residues in the biological activity, underlining the relevance of several residues involved in membrane interaction and the low correlation between stability and activity and (b) three amino acids involved in maturation, providing information on the specificity of the leader peptidase and the circularization process itself. To investigate the role of circularity in the stability and biological properties of the enterocin AS-48, two different ways of linearization have been attempted: in vitro by limited proteolysis experiments and in vivo by circular permutation in the structural gene as-48A. The results summarized here show the significance of circularization on the secondary structure, potency and, especially, the stability of AS-48 and point as well to a putative role of the leader peptide as a protecting moiety in the pre-proprotein. Taken all together, the data available on circular bacteriocins support the idea that AS-48 has been engineered by nature to make a remarkably active and stable protein with a broad spectrum of activity.
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