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Article
Function of the Signal Peptide and N- and C-terminal Propeptides in the Leucine Aminopeptidase from Aeromonas proteolytica
Protein Expression and Purification
  • Krzysztof P. Bzymek, Utah State University
  • Ventris M. D'Souza, Utah State University
  • Guanjing Chen, Utah State University
  • Heidi Campbell, Utah State University
  • Alice Mitchell, Utah State University
  • Richard C. Holz, Marquette University
Document Type
Article
Publication Date
10-1-2004
Disciplines
Abstract
The leucine aminopeptidase from Aeromonas proteolytica (also known as Vibrio proteolyticus) (AAP) is a metalloenzyme with broad substrate specificity. The open reading frame (ORF) for AAP encodes a 54 kDa enzyme, however, the extracellular enzyme has a molecular weight of 43 kDa. This form of AAP is further processed to a mature, thermostable 32 kDa form but the exact nature of this process is unknown. Over-expression of different forms of AAP in Escherichia coli (with AAP's native leader sequence, with and without the N- and/or C-terminal propeptides, and as fusion protein) has allowed a model for the processing of wild-type AAP to be proposed. The role of the A. proteolytica signal peptide in protein secretion as well as comparison to other known signal peptides reveals a close resemblance of the A. proteolytica signal peptide to the outer membrane protein (OmpA) signal peptide. Over-expression of the full 54 kDa AAP enzyme provides an enzyme that is significantly less active, due to a cooperative inhibitory interaction between both propeptides. Over-expression of AAP lacking its C-terminal propeptide provided an enzyme with an identical kcat value to wild-type AAP but exhibited a larger Km value, suggesting competitive inhibition of AAP by the N-terminal propeptide (Ki∼0.13 nM). The recombinant 32 kDa form of AAP was characterized by kinetic and spectroscopic methods and was shown to be identical to mature, wild-type AAP. Therefore, the ease of purification and processing of rAAP along with the fact that large quantities can be obtained now allow new detailed mechanistic studies to be performed on AAP through site-directed mutagenesis.
Comments

Protein Expression and Purification, Vol. 37, No. 2 (October 2004): 294-305. DOI.

Richard Holz was affiliated with the Utah State University at the time of publication.

Citation Information
Krzysztof P. Bzymek, Ventris M. D'Souza, Guanjing Chen, Heidi Campbell, et al.. "Function of the Signal Peptide and N- and C-terminal Propeptides in the Leucine Aminopeptidase from Aeromonas proteolytica" Protein Expression and Purification (2004) ISSN: 1046-5928
Available at: http://works.bepress.com/richard_holz/35/