Function of the signal peptide and N- and C-terminal propeptides in the leucine aminopeptidase from Aeromonas proteolytica

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 approximately 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.

Zinc Coordination Geometry and Ligand Binding Affinity: The Structural and Kinetic Analysis of the Second-Shell Serine 228 Residue and the Methionine 180 Residue of the Aminopeptidase from Vibrio proteolyticus

“…AAP is expressed with sequences that need to be proteolytically removed for full activity and crystallization. The protein was processed as previously reported (30). The mostly nonspecific endopeptidase, proteinase K, was added to the AAP solution to give a final concentration of 20 nM proteinase K, and the sample was incubated at 37 °C with mild rocking.…”

Heterologous expression and purification of Vibrio proteolyticus (Aeromonas proteolytica) aminopeptidase: A rapid protocol

“…The 54 kDa VpAP* polypeptide consists of a 21 amino acid signal peptide, an 85 amino acid N-terminal propeptide, a 299 amino acid mature region, and a 99 amino acid C-terminal propeptide (Figure 1). The signal peptide is required for secretion, the N-terminal propeptide is required for folding but its presence inhibits catalytic activity, and the C-terminal propeptide is of undetermined function but appears to be species-specific [71, 72]. VpAP* is processed to the final 32 kDa mature protein only after secretion and during purification; roles in the processing of VpAP* have been proposed for VpAP* itself (and VpAP) in autoprocessing, for a co-expressed neutral protease, and for a heat-treatment step that has been traditionally employed [73-75].…”

“…A highly detailed investigation into the requirements for the isolation of VpAP heterologously expressed in E. coli has been described by Bzymek, Holz and coworkers [71], and the resulting protocol has yielded both recombinant wild-type and site-directed variant forms of VpAP for subsequent crystallographic and physicochemical studies [38, 61-63, 71]. As suggested by earlier work [72, 77-79], it was found that VpAP was best expressed as a 52 kDa proenzyme (ΔspVpAP*; lacking the first 23 amino acids); the use of an appropriate leader sequence ( e.g.…”

“…In the course of our own studies, we have followed the earlier procedure [71] for the purification of recombinant wild-type and variant VpAP and have successfully isolated VpAP with amino acid substitutions in the active site pocket and in an adjacent hydrophobic pocket [39]. However, we have also encountered problems, including (i) irreproducible, and generally low, yields of VpAP; (ii) unpredictability of the effects of PK on variant forms of VpAP*; and (iii) the inability to isolate some site-directed variants as the mature proteins, notably variants with substitutions of Asp99.…”

Involvement of Propeptides in Formation of Catalytically Active Metalloproteinase from Thermoactinomyces sp.

“…The obtained data indicate the propeptide-assisted folding mechanism of the Mpr folding. In many proteases including thermolysin-like ones [1,3,22,[28][29][30], the N-terminal propeptide acts as an intramolecular chaperone in propeptide-assisted folding. However, our data provides the first demonstration that Mpr variants generated in cis and in trans differ in the ability to hydrolyze peptide substrates.…”