Cannabinoid biosynthesis using non-canonical enzymes

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Functional Evolution in Orthologous Cell-encoded RNA-dependent RNA Polymerases

Many eukaryotic organisms encode more than one RNA-dependent RNA polymerase (RdRP) that probably emerged as a result of gene duplication. Such RdRP paralogs often participate in distinct RNA silencing pathways and show characteristic repertoires of enzymatic activities in vitro. However, to what extent members of individual paralogous groups can undergo functional changes during speciation remains an open question. We show that orthologs of QDE-1, an RdRP component of the quelling pathway in Neurospora crassa, have rapidly diverged in evolution at the amino acid sequence level. Analyses of purified QDE-1 polymerases from N. crassa (QDE-1(Ncr)) and related fungi, Thielavia terrestris (QDE-1(Tte)) and Myceliophthora thermophila (QDE-1(Mth)), show that all three enzymes can synthesize RNA, but the precise modes of their action differ considerably. Unlike their QDE-1(Ncr) counterpart favoring processive RNA synthesis, QDE-1(Tte) and QDE-1(Mth) produce predominantly short RNA copies via primer-independent initiation. Surprisingly, a 3.19 Å resolution crystal structure of QDE-1(Tte) reveals a quasisymmetric dimer similar to QDE-1(Ncr). Further electron microscopy analyses confirm that QDE-1(Tte) occurs as a dimer in solution and retains this status upon interaction with a template. We conclude that divergence of orthologous RdRPs can result in functional innovation while retaining overall protein fold and quaternary structure

Turning an asparaginyl endopeptidase into a peptide ligase

Butelase-1 is a peptide asparaginyl ligase (PAL) that efficiently catalyzes peptide bond formation after Asn/Asp (Asx), whereas its homologue butelase-2 is an asparaginyl endopeptidase (AEP) that catalyzes the reverse reaction, hydrolyzing Asx-peptide bonds. Since PALs and AEPs share essentially similar overall structures, we surmised that the S2 and S1′ substrate-binding pockets immediately flanking the catalytic S1 site constitute the major ligase activity determinants (LADs) that control the catalytic directionality of these enzymes. Here, we report the successful conversion of butelase-2 into butelase-1-like ligases based on the LAD hypothesis. We prepared 23 LAD mutants by mutating residues of the S2 pocket (LAD1) and/or the S1′ pocket (LAD2) of butelase-2 into homologous residues in PALs. These LAD mutants markedly diminished protease activity and increased ligase activity. In contrast, substituting 12 non-LAD residues to the corresponding residues in butelase-1 did not change their protease profiles. At physiological pH, mutations targeting both LADs resulted in shifting the catalytic directionality from 95% hydrolysis to >95% peptide ligation. This results in the engineering of efficient recombinant peptide ligases that were demonstrated to be useful for macrocyclization and site-specific labeling of bioactive peptides and proteins. Five high-resolution crystal structures of butelase-2 and its engineered mutants reveal subtle changes proximal to the catalytic S1 site that account for the reversal in enzymatic activity. Computational simulations of enzyme-substrate complexes suggest how the S-acyl intermediate is positioned at the S2 site and the substrate orientated, controlling accessibility of either water or incoming nucleophiles from the prime-side (S1′ site) of the catalytic center. Together, these features determine the catalytic directionality between hydrolysis and ligation in AEPs and PALs. Overall, this work validates the LAD hypothesis as a central guide for making peptide ligases from their corresponding widely available protease counterparts, to produce precision tools for exquisite site-specific conjugation and the biomanufacturing of biologics.Ministry of Education (MOE)National Research Foundation (NRF)Accepted versionThis research was supported by the Ministry of Education, Singapore, under its MOE AcRF Tier 3 Award No. MOE2016- T3-1-003, NMRC Grant Nos.CBRG/0028/2014, NRF2016NRF-CRP001-063, and Synzymes and Natural Products Center (SYNC), Nanyang Technological Universit

Structural determinants for peptide-bond formation by asparaginyl ligases

Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptide-producing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1' sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1-3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs.Ministry of Education (MOE)Accepted versionThis research was supported by Academic Research Grant Tier 3 (MOE2016-T3-1-003) from the Singapore Ministry of Education (MOE) to the J.P.T., J.L., and C.-F.L. laboratories, and NMRC Grants CBRG/0028/2014 and NRF2016NRF-CRP001-063