O-methyltransferase-like enzyme catalyzed diazo installation in polyketide biosynthesis

Diazo compounds are rare natural products possessing various biological activities. Kinamycin and lomaiviticin, two diazo natural products featured by the diazobenzofluorene core, exhibit exceptional potency as chemotherapeutic agents. Despite the extensive studies on their biosynthetic gene clusters and the assembly of their polyketide scaffolds, the formation of the characteristic diazo group remains elusive. l-Glutamylhydrazine was recently shown to be the hydrazine donor in kinamycin biosynthesis, however, the mechanism for the installation of the hydrazine group onto the kinamycin scaffold is still unclear. Here we describe an O-methyltransferase-like protein, AlpH, which is responsible for the hydrazine incorporation in kinamycin biosynthesis. AlpH catalyses a unique SAM-independent coupling of l-glutamylhydrazine and polyketide intermediate via a rare Mannich reaction in polyketide biosynthesis. Our discovery expands the catalytic diversity of O-methyltransferase-like enzymes and lays a strong foundation for the discovery and development of novel diazo natural products through genome mining and synthetic biology.

. SDS-PAGE analysis of purified AlpJ, AlpK, and AlpH.The SDS-PAGE experiments were repeated tree times independently with similar results and the original photographs were supplied at the end of this file.Source data are provided as a Source Data file.

Supplementary Figure 4 .
In vitro characterization of Alp1W.(a) Purification of Alp1W: Lane1, whole cells; lane 2, supernatant; lane 3, flow through; lane 4, 30mM imidazole elution; lane 5, 500 mM imidazole elution.Source data are provided as a Source Data file.(b) HPLC analysis of the organic extracts of the enzymatic reactions.

6 .Supplementary
Supplementary Figure 12.HPLC analysis of the reactions with different combinations of enzymes and cofactors for the one-pot reaction.As AlpJ and Fre could catalyze the formation of 6 from DHR, Fre was used to replace AlpK to test the intermediacy of Figure 19.HPLC analysis of the competition reactions using different concentrations of NAC.DHR + AlpJ + AlpK + AlpH + NADH + gluN2H3 with 0 (i), 0.25 (ii), 1.25 (iii), and 2.5 (iv) mM NAC.
25mM):NAC(1.25mM)iii gluN 2 H 3 (0.25mM):NAC(0.25mM)ii iv Supplementary Figure 21.Structural comparison analyses of the overall structure of AlpH with the OMT LaPhzM and MmcR as well as the pericyclase PdxI.(a) Ribbon representation showing the overall structural comparison of AlpH and the SAM-dependent OMT LaPhzM in complex with SAH (PDB ID: 6C5B).In this drawing, AlpH is shown in orange and forest green, while LaPhzM is shown in grey and black.(b) Ribbon representation showing the overall structural comparison of the monomeric AlpH and the monomeric LaPhzM in complex with SAH (PDB ID: 6C5B).(c) Ribbon representation showing the overall structural comparison of AlpH and the SAM-dependent OMT MmcR in complex with SAH (PDB ID: 3GWZ).In this drawing, AlpH is shown in orange and forest green, while MmcR is shown in pink and violet.(d) Ribbon representation showing the overall structural comparison of the monomeric AlpH and the monomeric MmcR in complex with SAH (PDB ID: 3GWZ).(e) Ribbon representation showing the overall structural comparison of AlpH and the pericyclase PdxI (PDB ID: 7BQK).In this drawing, AlpH is shown in orange a AlpH dimer MmcR dimer (PDB ID: 3GWZ) , while PdxI is shown in light-blue and slate.(f) Ribbon representation showing the overall structural comparison of the monomeric AlpH and the monomeric PdxI (PDB ID: 7BQK).(GGGRG) motif of MmcR are also displayed, and the related hydrogen bonds involved in the binding are shown as dotted lines.(E) A structural modeling of AlpH with the co-factor SAH in the MmcR/SAH complex (PDB ID: 3GWZ).The relevant AlpH residues corresponding to the interface residues and the glycine-rich (GGGRG) motif of MmcR for interacting with SAH are shown in the stick-ball model.Notably, the side chain of AlpH Cys201 has a potential steric clash with the SAH molecule.

Table 2 .
Primers used in this study.

Table 3 .
Protein sequence homology of AlpH homologues.Σ|Fobs|, where Fobs and Fcalc are observed and calculated structure factors.Rfree = ΣT||Fobs| -|Fcalc||/ΣT|Fobs|, where T is a test data set of about 5% of the total reflections randomly chosen and set aside prior to refinement.Numbers in parentheses represent the value for the highest resolution shell.Supplementary Table6.1HNMR and 13 C NMR data for glutamylhydrazine in D2O.Supplementary Table7.1HNMRdatacomparison between reported prekinamycin and our isolated prekinamycin.Supplementary Table8.13CNMR data comparison between reported prekinamycin and our purified prekinamycin.