Endolysin enzymes from bacteriophage cause bacterial lysis by degrading the peptidoglycan cell wall. The streptococcal C1 phage endolysin PlyC, is the most potent endolysin described to date and can rapidly lyse group A, C, and E streptococci. PlyC Primer name Sequence (5′-3′) General sequencing fw seq TTAGCGGATCCTACCTGACG This study rv seq TTTTATCAGACCGCTTCTGC This study Site saturation mutagenesis and GA ins fw CAGTGCCAAAGAAACTGCTAAATGTTTTAG This study ins rev GGTTAGTTTGATAATGACACCATTCTAAGTTATG This study vector fw CATAACTTAGAATGGTGTCATTATCAAACTAACC This study vector rv CTAAAACATTTAGCAGTTTCTTTGGCACTG This study R66 NNS fw GATGTAGAGGCTATCNNSAAGGCTATGAAAAAG This study R66 NNS rv CTTTTCATAGCCTTNNSGATAGCCTCTACATC This study K59 NNS fw GTCTATCAATATTAGTNNSTCTGATGTAGAGGC This study K59 NNS rv GCCTCTACATCAGANNSACTAATATTGATAGAC This study Y28 NNS fw GAAAAGAAAGTTACGGTNNSCGTGCTTTTATTAACG This study Y28 NNS rv CGTTAATAAAAGCACGNNSACCGTAACTTTCTTTTC This study E36 NNS fw CTTTTATTAACGGAGTTNNSATTGGTATTAAAGACATTG This study E36 NNS rv CAATGTCTTTAATACCAATNNSAACTCCGTTAATAAAAG This study Y26 NNS fw CATACCGATGGAAAAGAAAGTNNSGGTTATCGTGCTTTTATTAAC This study Y26 NNS rv GTTAATAAAAGCACGATAACCNNSACTTTCTTTTCCATCGGTATG This study PlyCB site-directed mutagenesis PlyCB R66A fw GTAAGTCTGATGTAGAGGCTATCGCAAAGGCTATGAA This study PlyCB R66A rv TTCATAGCCTTTGCGATAGCCTCTACATCAGACTTAC This study PlyCB R66K fw CTGATGTAGAGGCTATCAAAAAGGCTATGAA This study PlyCB R66K rv TTCATAGCCTTTTTGATAGCCTCTACATCAG This study PlyCB Y26A fw GATGGAAAAGAAAGTGCCGGTTATCGTGCTTT This study PlyCB Y26A rv AAAGCACGATAACCGGCACTTTCTTTTCCATC This study PlyCB Y26W fw CGATGGAAAAGAAAGTTGGGGTTATCGTGCTTT This study PlyCB Y26W rv AAAGCACGATAACCCCAACTTTCTTTTCCATCG This study PlyCB Y28H fw AAGAAAGTTACGGTCATCGTGCTTTTATTAAC This study PlyCB Y28H rv GTTAATAAAAGCACGATGACCGTAACTTTCTT This study Note: All plasmids except pSM85 have a pBAD24 vector backbone and an ampicillin resistance marker. pSM85 is based on pBAD33 with chloramphenicol resistance.

During malarial infection, Plasmodium parasites digest human hemoglobin to obtain free amino acids for protein production and maintenance of osmotic pressure. The Plasmodium M1 and M17 aminopeptidases are both postulated to have an essential role in the terminal stages of the hemoglobin digestion process and are validated drug targets for the design of new dual-target anti-malarial compounds. In this study, we profiled the substrate specificity fingerprints and kinetic behaviors of M1 and M17 aminopeptidases from Plasmodium falciparum and Plasmodium vivax, and the mouse model species, Plasmodium berghei. We found that although the Plasmodium M1 aminopeptidases share a largely similar, broad specificity at the P1 position, the P. falciparum M1 displays the greatest diversity in specificity and P. berghei M1 showing a preference for charged P1 residues. In contrast, the Plasmodium M17 aminopeptidases share a highly conserved preference for hydrophobic residues at the P1 position. The aminopeptidases also demonstrated intra-peptide sequence specificity, particularly the M1 aminopeptidases, which showed a definitive preference for peptides with fewer negatively charged intrapeptide residues. Overall, the P. vivax and P. berghei enzymes had a faster substrate turnover rate than the P. falciparum enzymes, which we postulate is due to subtle differences in structural dynamicity. Together, these results build a kinetic profile that allows us to better understand the catalytic nuances of the M1 and M17 aminopeptidases from different Plasmodium species.

No abstract