Characterization of non-heme iron aliphatic halogenase WelO5* from Hapalosiphon welwitschii IC-52-3: Identification of a minimal protein sequence motif that confers enzymatic chlorination specificity in the biosynthesis of welwitindolelinones

Abstract

The in vitro biochemical characterization revealed that iron/2-oxoglutarate (Fe/2OG)-dependent aliphatic halogenase WelO5* in Hapalosiphon welwitschii IC-52-3 has an enhanced substrate specificity towards 12-epi-hapalindole C (1) in comparison to WelO5 in H. welwitschii UTEX B1830. This allowed us to define the origin of the varied chlorinated versus dechlorinated alkaloid structural diversity between the two welwitindolinone producers. Furthermore, this study, along with the recent characterization of the AmbO5 protein, collectively confirmed the presence of a signature sequence motif in the C-terminus of this newly discovered halogenase enzyme family that confers substrate promiscuity and specificity. These observations may guide the rational engineering and evolution of these proteins for biocatalyst application.

Evolved Aliphatic Halogenases Enable Regiocomplementary C−H Functionalization of a Pharmaceutically Relevant Compound

“…The recently discovered aliphatic halogenases AmbO5, WelO5, and WelO5* belong to the family of iron/α‐ketoglutarate (Fe/α‐KG)‐dependent oxygenases and, in contrast to the majority of members of this enzyme class, are capable of acting on freestanding substrates . These enzymes share an overall high sequence identity (>79 %), whereas WelO5 and WelO5* stem from different isolates of the same organism named Hapalosiphon welwitschii and differ only by 15 amino acids (Figure S1 in the Supporting Information) . In nature, these enzymes catalyze the installation of chlorine atoms into several complex indole alkaloids (Scheme ) .…”

Chlorinated Natural Products and Related Halogenases

“…A radical mechanism was proposed for SyrB2 and its crystal structure was reported ,. Similarly, formation of hapalindole‐type alkaloids was catalyzed by two nonheme iron‐dependent halogenases, WelO5 and AmbO5 . 12‐epi‐Hapalindole E, 12‐epi‐fischerindole G, and welwitindolinones A–C (Figure ) are all halogenated compounds catalyzed by iron‐α‐ketoglutarate‐dependent halogenases, though the radical mechanism has not been reported.…”

Discovery of a Calcium‐Dependent Enzymatic Cascade for the Selective Assembly of Hapalindole‐Type Alkaloids: On the Biosynthetic Origin of Hapalindole U

“…[1][2][3][4][5][6][7][8][9][10][11][12] These studies,c ollectively directed towards deciphering the origin of structural complexities and diversities of hapalindole-type alkaloids,h ave thus far unraveled several layers of puzzles concerning their global biosynthetic diversifications (see Figure S1 in the Supporting Information). [1][2][3][4][5][6][7][8][9][10][11][12] These studies,c ollectively directed towards deciphering the origin of structural complexities and diversities of hapalindole-type alkaloids,h ave thus far unraveled several layers of puzzles concerning their global biosynthetic diversifications (see Figure S1 in the Supporting Information).…”

“…[1][2][3][4][5][6][7][8][9][10][11][12] These studies,c ollectively directed towards deciphering the origin of structural complexities and diversities of hapalindole-type alkaloids,h ave thus far unraveled several layers of puzzles concerning their global biosynthetic diversifications (see Figure S1 in the Supporting Information). [4,6,12] More recently,anew family of proteins embedded in the hapalindole-type alkaloid biosynthetic pathways,originally named as U-proteins for their bioinformatically unknown nature, [1][2][3] were shown able to stereoselectively rearrange and cyclize 2 to three isomeric hapalindole-type alkaloids,t ypified by 12epi-fischerindole Us ynthase WelU1, 12-epi-hapalindole C synthase WelU3 and 12-epi-hapalindole Us ynthase AmbU4. [4,6,12] More recently,anew family of proteins embedded in the hapalindole-type alkaloid biosynthetic pathways,originally named as U-proteins for their bioinformatically unknown nature, [1][2][3] were shown able to stereoselectively rearrange and cyclize 2 to three isomeric hapalindole-type alkaloids,t ypified by 12epi-fischerindole Us ynthase WelU1, 12-epi-hapalindole C synthase WelU3 and 12-epi-hapalindole Us ynthase AmbU4.…”

“…[10,11] As the enzymatic cyclizations by WelU1/ WelU3/AmbU4 proteins are induced by the geranyl group rearrangement in 2,these enzymes can be formally named as afamily of isomerocyclases. [11,12] In contrast to the welwitindolinones,the enzymatic pathways for the assembly of ambiguine precursors remain incomplete.W er ecently showed that hapalindole U 4 is ab iosynthetic precursor to hapalindole G 5 and related ambiguines in Fischerella ambigua UTEX1903 (Figures 1b and S2) by characterizing the pathway-specific prenyltransferase AmbP3 and halogenase AmbO5 (Figure 1c and S3). [11,12] In contrast to the welwitindolinones,the enzymatic pathways for the assembly of ambiguine precursors remain incomplete.W er ecently showed that hapalindole U 4 is ab iosynthetic precursor to hapalindole G 5 and related ambiguines in Fischerella ambigua UTEX1903 (Figures 1b and S2) by characterizing the pathway-specific prenyltransferase AmbP3 and halogenase AmbO5 (Figure 1c and S3).…”