7 research outputs found
Mechanistic Studies on a Remarkable Flavoenzyme Involved in Roseoflavin Biosynthesis
Roseoflavin is a bright red-colored anti-vitamin biosynthesized by Streptomyces davawensis and Streptomyces cinnabarinus. It is the only naturally occurring riboflavin analog that exhibits antibiotic properties. Earlier studies show that roseoflavin is biosynthesized from riboflavin by substitution of the C8 methyl with a dimethylamino group1. Recently, the methyltransferase catalyzing the final step has been characterized and a sub-genomic fragment containing genes sufficient for the entire biosynthesis has also been cloned. However, the mechanism of biosynthesis of the key intermediate 8-amino-flavin is poorly understood. Herein, we report the identification and characterization of a single flavin-dependent enzyme, RosB that replaces the C8 methyl group of Flavin mononucleotide (FMN) and uses the amino group of glutamate to convert FMN to 8-amino-FMN via the intermediacy of 8-formyl-FMN. The enzyme was cloned, overexpressed and purified, and subsequently tested for the predicted activity. A novel mechanism for the RosB-catalyzed reaction is proposed based on the identification of reaction products and intermediates. In the first step of this reaction, the enzyme oxidizes the C8 methyl group of FMN to a formyl group using molecular oxygen. Next, the intermediate, 8-formyl-FMN catalyzes the transfer of the amino group from the glutamate to flavin. The resulting reactive FMN intermediate then rearranges to yield 8-amino-FMN and the C8 methyl group is lost as formate. To gain further insights into this unprecedented amine transfer reaction various substrate analogs were designed and studied. Reactive intermediates were successfully trapped and characterized by HPLC and LCMS studies. Additionally, the crystal structure of the enzyme containing a bound inhibitor complemented the results of the biochemical analysis. Overall, this work reveals the existence of yet another novel flavin transformation involved in natural product biosynthesis. Moreover, we identified and characterized the phosphatase, RosC that converts 8-amino FMN, the product of RosB to 8-amino riboflavin, the substrate of the next enzyme in roseoflavin biosynthesis pathway. Thus, identification of RosB and RosC completed the biosynthetic pathway of roseoflavin. These studies demonstrated the power of mechanism guided approach to identify enzymes involved in natural product biosynthesis pathway
Mechanistic Studies on a Remarkable Flavoenzyme Involved in Roseoflavin Biosynthesis
Roseoflavin is a bright red-colored anti-vitamin biosynthesized by Streptomyces davawensis and Streptomyces cinnabarinus. It is the only naturally occurring riboflavin analog that exhibits antibiotic properties. Earlier studies show that roseoflavin is biosynthesized from riboflavin by substitution of the C8 methyl with a dimethylamino group1. Recently, the methyltransferase catalyzing the final step has been characterized and a sub-genomic fragment containing genes sufficient for the entire biosynthesis has also been cloned. However, the mechanism of biosynthesis of the key intermediate 8-amino-flavin is poorly understood. Herein, we report the identification and characterization of a single flavin-dependent enzyme, RosB that replaces the C8 methyl group of Flavin mononucleotide (FMN) and uses the amino group of glutamate to convert FMN to 8-amino-FMN via the intermediacy of 8-formyl-FMN. The enzyme was cloned, overexpressed and purified, and subsequently tested for the predicted activity. A novel mechanism for the RosB-catalyzed reaction is proposed based on the identification of reaction products and intermediates. In the first step of this reaction, the enzyme oxidizes the C8 methyl group of FMN to a formyl group using molecular oxygen. Next, the intermediate, 8-formyl-FMN catalyzes the transfer of the amino group from the glutamate to flavin. The resulting reactive FMN intermediate then rearranges to yield 8-amino-FMN and the C8 methyl group is lost as formate. To gain further insights into this unprecedented amine transfer reaction various substrate analogs were designed and studied. Reactive intermediates were successfully trapped and characterized by HPLC and LCMS studies. Additionally, the crystal structure of the enzyme containing a bound inhibitor complemented the results of the biochemical analysis. Overall, this work reveals the existence of yet another novel flavin transformation involved in natural product biosynthesis. Moreover, we identified and characterized the phosphatase, RosC that converts 8-amino FMN, the product of RosB to 8-amino riboflavin, the substrate of the next enzyme in roseoflavin biosynthesis pathway. Thus, identification of RosB and RosC completed the biosynthetic pathway of roseoflavin. These studies demonstrated the power of mechanism guided approach to identify enzymes involved in natural product biosynthesis pathway
A Remarkable Oxidative Cascade That Replaces the Riboflavin C8 Methyl with an Amino Group during Roseoflavin Biosynthesis
Roseoflavin is a naturally occurring riboflavin analogue with antibiotic properties. It is biosynthesized from riboflavin in a reaction involving replacement of the C8 methyl with a dimethylamino group. Herein we report the identification of a flavin-dependent enzyme that converts flavin mononucleotide (FMN) and glutamate to 8-amino-FMN via the intermediacy of 8-formyl-FMN. A mechanistic proposal for this remarkable transformation is proposed
A Remarkable Oxidative Cascade That Replaces the Riboflavin C8 Methyl with an Amino Group during Roseoflavin Biosynthesis
Roseoflavin
is a naturally occurring riboflavin analogue with antibiotic
properties. It is biosynthesized from riboflavin in a reaction involving
replacement of the C8 methyl with a dimethylamino group. Herein we
report the identification of a flavin-dependent enzyme that converts
flavin mononucleotide (FMN) and glutamate to 8-amino-FMN via the intermediacy
of 8-formyl-FMN. A mechanistic proposal for this remarkable transformation
is proposed
A Remarkable Oxidative Cascade That Replaces the Riboflavin C8 Methyl with an Amino Group during Roseoflavin Biosynthesis
Roseoflavin
is a naturally occurring riboflavin analogue with antibiotic
properties. It is biosynthesized from riboflavin in a reaction involving
replacement of the C8 methyl with a dimethylamino group. Herein we
report the identification of a flavin-dependent enzyme that converts
flavin mononucleotide (FMN) and glutamate to 8-amino-FMN via the intermediacy
of 8-formyl-FMN. A mechanistic proposal for this remarkable transformation
is proposed
Radical S-adenosylmethionine (SAM) enzymes in cofactor biosynthesis: A treasure trove of complex organic radical rearrangement reactions
In this minireview, we describe the radical S-adenosylmethionine enzymes involved in the biosynthesis of thiamin, menaquinone, molybdopterin, coenzyme F420, and heme. Our focus is on the remarkably complex organic rearrangements involved, many of which have no precedent in organic or biological chemistry