Enzymes of nikkomycin biosynthesis

Nikkomycins are produced by several species of Streptomyces and exhibit fungicidal, insecticidal and acaricidal properties due to their strong inhibition of chitin synthase. Since chitin biosynthesis is vital for the integrity of the cell wall of fungi and the exoskeleton of insects, spiders and other invertebrates, nikkomycins have potential as antibiotics. Structurally, nikkomycins are peptidyl nucleosides containing two unusual amino acids, i.e. hydroxypyridylhomothreonine and aminohexuronic acid with either uracil or 4-formyl-4-imidazolin-2-one as N- glycosidically linked bases. Although the chemical structures of nikkomycins have been known since the 1970s, only a few biosynthetic steps have been elucidated. Especially the enzymatic reactions leading to the aminohexuronic acid moiety are elusive. We have recently shown that a protein encoded by nikO catalyzes the transfer of enolpyruvyl to the 3`-hydroxyl group of UMP. Currently, nothing is known about the enzymes and the putative chemical steps that are involved in the transformations following this initial reaction. However, the genes that are co-transcribed with nikO, designated nikI, nikJ, nikK, nikL, nikM and nikN, encode for enzymes that can be expected to catalyze the reactions required to transform 3`-enolpyruvyl-UMP to the aminohexuronic acid moiety. In this research proposal, we propose to identify the genes that are involved in the transformation of 3`- enolpyruvyl-UMP to the putative nikkomycin intermediates "S" (designated SX and SZ depending on the base attached to the sugar moiety). In our working hypothesis we propose that dephosphorylation and cyclisation of 3`- enolpyruvyl-UMP generates intermediate "S", which is then converted to the aminohexuronic acid moiety. Following the identification of the genes encoding for the putative phosphatase and "cyclase", we will iniatiate the cloning and heterologous expression to obtain sufficient quantitities of the proteins to make them available for biochemical studies. In addition to the biochemical characterization of the enzymes, the proteins will be crystallized in order to determine their three-dimensional structures. Through this combined biochemical and structural approach, we will be able to develop a detailed understanding of the structure-function relationships that govern enzyme catalysis and selection of substrates. This research proposal sets the stage towards the elucidation of the entire pathway leading to the generation of the aminohexuronic acid moiety. As such, it will be an important achievement in the understanding of nikkomycin biosynthesis and it will enable us to design strategies for the synthesis of new nikkomycin compounds, e.g. by mutasynthesis, that may be of potential use as antibiotics.

Nikkomycins are produced by several species of Streptomyces and exhibit fungicidal, insecticidal and acaricidal properties due to their strong inhibition of chitin synthase. Since chitin biosynthesis is vital for the integrity of the cell wall of fungi and the exoskeleton of insects, spiders and other invertebrates, nikkomycins have potential as antibiotics. Structurally, nikkomycins are peptidyl nucleosides containing two unusual amino acids, i.e. hydroxypyridylhomothreonine and aminohexuronic acid with either uracil or 4-formyl-4-imidazolin-2-one as N- glycosidically linked bases. Although the chemical structures of nikkomycins have been known since the 1970s, only a few biosynthetic steps have been elucidated. Especially the enzymatic reactions leading to the aminohexuronic acid moiety are elusive. We have recently shown that a protein encoded by nikO catalyzes the transfer of enolpyruvyl to the 3`-hydroxyl group of UMP. Currently, nothing is known about the enzymes and the putative chemical steps that are involved in the transformations following this initial reaction. However, the genes that are co-transcribed with nikO, designated nikI, nikJ, nikK, nikL, nikM and nikN, encode for enzymes that can be expected to catalyze the reactions required to transform 3`-enolpyruvyl-UMP to the aminohexuronic acid moiety. In this research proposal, we propose to identify the genes that are involved in the transformation of 3`- enolpyruvyl-UMP to the putative nikkomycin intermediates "S" (designated SX and SZ depending on the base attached to the sugar moiety). In our working hypothesis we propose that dephosphorylation and cyclisation of 3`- enolpyruvyl-UMP generates intermediate "S", which is then converted to the aminohexuronic acid moiety. Following the identification of the genes encoding for the putative phosphatase and "cyclase", we will iniatiate the cloning and heterologous expression to obtain sufficient quantitities of the proteins to make them available for biochemical studies. In addition to the biochemical characterization of the enzymes, the proteins will be crystallized in order to determine their three-dimensional structures. Through this combined biochemical and structural approach, we will be able to develop a detailed understanding of the structure-function relationships that govern enzyme catalysis and selection of substrates. This research proposal sets the stage towards the elucidation of the entire pathway leading to the generation of the aminohexuronic acid moiety. As such, it will be an important achievement in the understanding of nikkomycin biosynthesis and it will enable us to design strategies for the synthesis of new nikkomycin compounds, e.g. by mutasynthesis, that may be of potential use as antibiotics.