Heterologous expression of peptidyl-Lys metallopeptidase ofArmillaria melleaand mutagenic analysis of the recombinant peptidase

Abstract

A method to express, purify and modify the PeptidylLys metallopeptidase (LysN) of Armillaria mellea in Pichia pastoris was developed to enable functional studies of the protease. Based on prior work, we propose a mechanism of action of LysN. Catalytic residues were investigated by site-directed mutagenesis. As anticipated, these mutations resulted in significantly reduced catalytic rates. Additionally, based on molecular modelling eleven mutants were designed to have altered substrate specificity. The S 1 0 binding pocket of LysN is quite narrow and lined with negative charge to specifically accommodate lysine. To allow for arginine specificity in S 1 0 , it was proposed to extend the S 1 0 binding pocket by mutagenesis, however the resulting mutant did not show any activity with arginine in P 1 0 . Two mutants, A101D and T105D, showed increased specificity towards arginine in subsites S 2 0 S 4 0 compared to the wild type protease. We speculate that the increased specificity to result from the additional negative charge which attract and interact with positively charged residues better than the wild type.Keywords: enzyme kinetics/enzyme mechanism/ metalloprotease/molecular modelling/site-directed mutagenesis.Abbreviations: Am-LysN, Armillaria mellea peptidyl-Lys metallopeptidase; DsbA, disulfideoxidoreductase; FRET, Fo¨rster resonance energy transfer; GAP, glyceraldehyde-3-phosphatedehydrogenase; IPTG, isopropylb-D-1-thiogalactopyranoside; MOE; Molecular Operation Environment (CCG); MOPS, 3-(N-morpholine)propanesulfonic acid; MS, mass spectrometry; PCR, polymerase chain reaction; RMSD, root-mean-square deviation; SDSPAGE, sodiumdodecylsulfatepolyacrylamide gel electrophoresis.In this article, we use Schechter and Berger nomenclature (1).Peptidyl-Lys metallopeptidase (LysN) from Armillaria mellea is an aspzincin metalloprotease, which cleaves in front of lysines (2). Hence, it is unusual by having its substrate specificity at the C-terminal side of the scissile bond (/K) (3). The protease is predominantly used as a tool in proteomic studies, where it serves as an alternative or complement to trypsin. The advantage of LysN is that it generates fragments which have a positive charge at the N-terminus, yielding mass spectrometry (MS) fragmentation with clear b-ion ladders (4,5). This feature can be used to facilitate de novo peptide mapping. Recently we profiled the substrate specificity of AmLysN and found it to be strictly lysine specific at S 1 0 , and having decreasing specificity when moving away from the scissile bond (2, 6).We also reported a homology model of Am-LysN based on the crystal structure of Gf-LysN (7). The two structures had a RMSD of 0.001 on backbone residues and 0.239 on the side chain residues (2). In this model Am-LysN has a narrow, and relatively short (57 amino acid) binding cleft. Central to the hydrolysis of peptides, is the zinc ion and the zinc binding motif 'HExxH + D'. Fushimi et al. showed that the aspartic acid D132 is necessary to coordinate zinc binding of the homologous protein ...

A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii

“…The observed MW of Tc -LysN is consistent with the MWs of previously studied LysN peptidases (Wingard et al 1972 ; Nonaka et al 1995 ; Saito et al 2002 ; Stressler et al 2014 ; Ødum et al 2016 a). The Tc -LysN activity obtained at the end of purification was ~40 ACU under optimal conditions, which corresponded to an activity yield of ~9%.…”

“…Due to the unavailability of data about enzyme activities of recombinant LysN peptidases reported in other studies, a comparison between enzyme activities could not be made. The concentration of the purified Tc -LysN was ~1.3 mg*L −1 , which is 5.2 times higher than the hexa-histidine-tagged recombinant Am -LysN expressed in K. phaffii (Ødum et al 2016 ) . The use of a 5 kDa membrane for cross-flow filtration during downstream processing could have resulted in some loss of the ~19.8 kDa Tc- LysN resulting in the reduced final yield.…”

“…Even small changes in the pro-peptide sequence can have a consequential impact on the expression and activity of the recombinant peptidase (Boon et al 2020 ). Higher expression levels of recombinant Am -LysN were reported by Ødum et al ( 2016 ) when native pro-peptide sequence was used.…”

“…Except for Gf -LysN reported by Stressler et al ( 2014 ), which had a temperature maximum of 55 °C, explicit data about temperature maxima for the other LysN peptidases could not be found. All LysN peptidases, however, were reported to be pH and temperature stable (Lewis et al 1978 ; Nonaka et al 1995 ; Nonaka et al 1998 ; Ødum et al 2016 ; Wingard et al 1972 ). Tc -LysN demonstrated maximum proteolytic activity at 60 °C, while maintaining a wide operational range between 40 °C—70 °C (>50% proteolytic activity).…”

“…These enzymes were also found to be quite thermostable and exhibited relatively high resistance to denaturants such as urea and guanidine hydrochloride. Gf -LysN (Saito et al 2002 ) and Am -LysN (Ødum et al 2016 ) have also been recombinantly expressed in Komagataella phaffii , both as inactive protein (zymogen) and mature (active) protein using the full-length native pre-pro-protein, pro-protein, and mature protein coding sequences.…”