The role of propeptide-mediated autoinhibition and intermolecular chaperone in the maturation of cognate catalytic domain in leucine aminopeptidase

https://doi.org/10.1016/j.jsb.2021.107741Get rights and content

Highlights

  • AO-proLapA and AO-mLapA are novel fungal M28 aminopeptidase structures.

  • AO-LapA propeptide acts as intramolecular chaperone.

  • Structures reveal molecular mechanism of AO-LapA catalytic domain autoinhibition.

  • Polar contacts between propeptide and catalytic domain mimic inhibitor interactions.

Abstract

Leucyl aminopeptidase A from Aspergillus oryzae RIB40 (AO-LapA) is an exo-acting peptidase, widely utilised in food debittering applications. AO-LapA is secreted as a zymogen by the host and requires enzymatic cleavage of the autoinhibitory propeptide to reveal its full activity. Scarcity of structural data of zymogen aminopeptidases hampers a better understanding of the details of their molecular action of autoinhibition and how this might be utilised to improve the properties of such enzymes by recombinant methods for more effective bioprocessing. To address this gap in the literature, herein we report high-resolution crystal structures of recombinantly expressed AO-LapA precursor (AO-proLapA), mature LapA (AO-mLapA) and AO-mLapA complexed with reaction product l-leucine (AO-mLapA-Leu), all purified from Pichia pastoris culture supernatant. Our structures reveal a plausible molecular mechanism of LapA catalytic domain autoinhibition by propeptide and highlights the role of intramolecular chaperone (IMC). Our data suggest an absolute requirement for IMC in the maturation of cognate catalytic domain of AO-LapA. This observation is reinforced by our expression and refolding data of catalytic domain only (AO-refLapA) from Escherichia coli inclusion bodies, revealing a limited active conformation. Our work supports the notion that known synthetic aminopeptidase inhibitors and substrates mimic key polar contacts between propeptide and corresponding catalytic domain, demonstrated in our AO-proLapA zymogen crystal structure. Furthermore, understanding the atomic details of the autoinhibitory mechanism of cognate catalytic domains by native propeptides has wider reaching implications toward synthetic production of more effective inhibitors of bimetallic aminopeptidases and other dizinc enzymes that share an analogous reaction mechanism.

Introduction

Aminopeptidases are utilised in the food industry for sensory applications and development of food flavour profile. Aminopeptidases are indispensable in protein hydrolysate debittering, since they remove hydrophobic amino acids from the N-terminus of peptide hydrolysates, which contribute to bitterness of the flavour profile (Stressler et al., 2013, Nampoothiri et al., 2005, Lin et al., 2004). Fungal aminopeptidases from Aspergillus oryzae are generally recognized as safe (GRAS) by the food industry, with a long history of utilisation in debittering applications. Efforts to characterise their role in this process, led to earlier work to over-express and biochemically characterize the extracellular leucine aminopeptidase A from A. oryzae RIB40 (AO-LapA) (Matsushita-Morita et al., 2011). To date, no additional reports of exogenous over-expression of this high-utility aminopeptidase exist in the literature. Also lacking are attempts of successful production of such peptidases in Pichia pastoris, an important GRAS organism used in the food industry.

To our knowledge, there have been no published data for the crystal structures of the leucine aminopeptidases used in the food industry. The scarcity of precise structural data for these predominantly fungal peptidases inhibits our understanding of their reaction-inhibition mechanisms and substrate specificities. For example, there are only 12 amino acid differences between the full sequences of AO-LapA and AS-Lap1 (leucine aminopeptidase from Aspergillus sojae), which mostly occur outside the active site pocket, that is responsible for their quite distinct substrate preferences (Huang et al., 2015).

It is well established, that pro-domains of secretory endo- and exopeptidases play an important role in the inhibition of their cognate catalytic domains, which undergo activation by removal of the pro-domain either by autocatalytic processing or external endopeptidase activity. By secreting protease precursors in a zymogen form, undesirable cytosolic activation and proteolysis events are prevented (Lazure, 2002). Typically, the zymogen conversion does not involve conformational changes of catalytic residues and the active sites are sterically rendered inaccessible to substrate by the unique residues of pro-domain or prosegment, thus preventing activity (Khan et al., 1999). In addition, N-terminal propeptides are known to act as intramolecular chaperones (IMC), decreasing the energy barrier of transition from a molten globule intermediate to a kinetically trapped native state (Subbian et al., 2015). In this respect, a well-studied model enzyme, subtilisin, has been shown to be unable to escape the folding transition state between a molten-globule and a native conformation, without the presence of a pro-domain; a rate-limiting step in the folding process of subtilisin (Shinde and Inouye, 2000). Remarkably, pro-domains of numerous proteinases have been reported to assist as chaperones in both inter- and intramolecular manner, following expression in trans- and cis- form, respectively (Beggah et al., 2000, Tang et al., 2002, Safina et al., 2011). An analogous mode of action for such pro-domains, with no requirement for a covalent link between propeptide and catalytic domain, has been discovered in other enzymes (Yurimoto et al., 2004).

Indeed, both subtle and rather complex relationships between propeptide and catalytic domain were found in subtilisin E, where an unaltered, mature polypeptide chain of subtilisin E was shown to fold into a different conformation via a mutated intramolecular chaperone and to show altered substrate specificity; a phenomenon termed ‘protein memory‘ (Shinde et al., 1997). Normally, such intramolecular chaperones precede catalytic domain from the N-terminus, however, eukaryotic monozinc aminopeptidase A was found to recruit its C-terminal propeptide for this role (Rozenfeld et al., 2004). Such findings demonstrate the diverse and complex relationships between the essential pro-domain and the cognate active conformation in the proteogenesis of proteases.

The aminopeptidase from Vibrio proteolyticus (AAP) is a model bimetallohydrolase enzyme, representing the M28 peptidase family (MH clan) whose extensive structural and functional studies have provided an in-depth understanding of the mechanism of action and the substrate preferences for these bi-metallic enzymes (Schalk et al., 1992, Chevrier et al., 1994, Chevrier et al., 1996, Bennett and Holz, 1997, Chen et al., 1997, Stamper et al., 2001, Stamper et al., 2004, Schurer et al., 2004, Desmarais et al., 2006, Kumar et al., 2007). Furthermore, the role of the propeptide in AAP and other homologous leucine aminopeptidases has been extensively studied in vitro (Zhang et al., 2000, Bzymek et al., 2004, Nirasawa et al., 1999). A number of studies have provided structural insights into AAP inhibition via different synthetic compounds, acting as competitive inhibitors (Chevrier et al., 1996, Stamper et al., 2001, Stamper et al., 2004, Hanaya et al., 2012). However, until now, no report exists to provide a detailed mechanistic insight for leucine aminopeptidase inhibition by its natural competitive inhibitor – the N-terminal propeptide - at the molecular level.

In this article, we present the crystal structures of recombinantly expressed LapA precursor (AO-proLapA), mature LapA (AO-mLapA) and mature form of the enzyme complexed with reaction product l-leucine (AO-mLapA-Leu), all purified from P. pastoris culture supernatant. To date, the exact mechanism of leucine aminopeptidase inhibition and the role of an intramolecular chaperone (IMC) by the pro-domain has not been described in detail. To the best of our knowledge, this is the first crystal structure of a leucine aminopeptidase precursor, revealing detailed structural aspects of catalytic domain inhibition by the cognate propeptide. This work shows that known synthetic aminopeptidase inhibitors and substrates mimic key polar contacts between propeptide and the catalytic domain. These findings could aid the future design of more effective inhibitors of bimetallic aminopeptidases and other dizinc enzymes, sharing a similar reaction mechanism.

Section snippets

Sequence comparison of Lap peptidases

Extracellular aminopeptidases from the M28 family share a very similar sequence composition of their open reading frames (ORF‘s). In general, a short secretion signal sequence is preceded by a moderate length propeptide (removed in the maturation process), which defines a central catalytic domain. It is convenient to compare their primary sequences and their tertiary structure folds to establish conservation and evolutionary divergence at both the sequence and structure level. Evolutionary

Conclusion

In this work, the role of propeptide in the expression construct of LapA precursor was investigated, using a P. pastoris recombinant expression platform. Due to the propeptide deletion AO-ΔproLapA, recombinant LapA was secreted at significantly reduced levels compared to full LapA precursor (AO-proLapA). Lack of the propeptide in AO-ΔproLapA expression cassette could have yielded inappropriately folded protein with exposed hydrophobic patches, associating with molecular chaperones, the

Strains and plasmids

P. pastoris BG10 strain was obtained from Biogrammatics Inc. (Carlsbad, CA, USA). Secretory expression vector, pJ902, was provided by DNA 2.0 (USA). E. coli DH5α (fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17) cloning strain was purchased from New England Biolabs (NEB,UK).

All primers used in this study were obtained from Sigma (UK). ZeocinTM was purchased from Invitrogen (USA). All other reagents were purchased from Sigma (UK) and were of analytical

Accession numbers

Structural data were deposited in the PDB database with the following accession codes: 6ZEP (AO-proLapA), 6ZEQ (AO-mLapA) and 7OEZ (AO-mLapA-Leu).

CRediT authorship contribution statement

G. Baltulionis: Conceptualization, Investigation, Methodology, Formal analysis, Visualization, Writing - original draft, Writing - review & editing. M. Blight: Conceptualization, Resources, Methodology. A. Robin: Conceptualization, Resources, Methodology. D. Charalampopoulos: Funding acquisition, Conceptualization, Resources, Supervision, Writing - review & editing. K.A. Watson: Funding acquisition, Conceptualization, Resources, Methodology, Formal analysis, Visualization, Supervision, Writing

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank Diamond Light Source, University of Reading and Biocatalysts Ltd. for access to excellent facilities. This work was funded by a BBSRC CASE award BB/K012053/1 (to GB).

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