Construction of the Octose 8-Phosphate Intermediate in Lincomycin A Biosynthesis: Characterization of the Reactions Catalyzed by LmbR and LmbN

Abstract

Lincomycin A is a potent antimicrobial agent noted for its unusual C1 methylmercapto-substituted eight-carbon sugar. Despite its long clinical history for the treatment of Grampositive infections, the biosynthesis of the C8-sugar, methylthiolincosamide (MTL), is poorly understood. Here, we report our studies of the two initial enzymatic steps in the MTL biosynthetic pathway leading to the identification of D-erythro-D-gluco-octose 8-phosphate as a key intermediate. Our experiments demonstrate that this intermediate is formed via a transaldol reaction catalyzed by LmbR using D-fructose 6-phosphate or D-sedoheptulose 7-phosphate as the C3 donor and D-ribose 5-phosphate as the C5 acceptor. Subsequent 1,2-isomerization catalyzed by LmbN converts the resulting 2-keto C8-sugar (octulose 8-phosphate) to octose 8-phosphate. These results provide, for the first time, in vitro evidence revealing the biosynthetic origin of the C8 backbone of MTL.

The Novel Transcriptional Regulator LmbU Promotes Lincomycin Biosynthesis through Regulating Expression of Its Target Genes in Streptomyces lincolnensis

“…The MTL pathway is not yet well understood. Five genes (lmbR, lmbN, lmbP, lmbO, and lmbK) were identified to be involved in MTL biosynthesis (15,16) (Fig. 1B).…”

“…On the other hand, it was proposed that MTL is derived from the pentose phosphate cycle (15,16). In the early stages of MTL biosynthesis, the LmbR transaldolase catalyzes a transaldol reaction to produce the C8 sugar octulose 8-phosphate, which is subsequently isomerized to octose 8-phosphate by LmbN (15).…”

“…In the early stages of MTL biosynthesis, the LmbR transaldolase catalyzes a transaldol reaction to produce the C8 sugar octulose 8-phosphate, which is subsequently isomerized to octose 8-phosphate by LmbN (15). Then, the putative LmbP kinase phosphorylates the octose 8-phosphate to octose 1,8-bisphosphate.…”

In Vitro Characterization of LmbK and LmbO: Identification of GDP-d-erythro-α-d-gluco-octose as a Key Intermediate in Lincomycin A Biosynthesis

“…12 Subsequent isomerization catalyzed by LmbN converts the resulting octulose 8-phosphate ( 7 ) to octose 8-phosphate ( 8 ). 12 The fact that several genes homologous to those found in various NDP-deoxyhexose pathways 13 exist in the lincomycin gene cluster (designated as lmb ) prompted us to hypothesize that an NDP-activated octopyranose is a possible intermediate in the MTL ( 3 ) biosynthetic pathway. 12 However, this hypothesis lacks literature precedence because NDP-octose has never been reported to be an biosynthetic intermediate.…”

“…12 The fact that several genes homologous to those found in various NDP-deoxyhexose pathways 13 exist in the lincomycin gene cluster (designated as lmb ) prompted us to hypothesize that an NDP-activated octopyranose is a possible intermediate in the MTL ( 3 ) biosynthetic pathway. 12 However, this hypothesis lacks literature precedence because NDP-octose has never been reported to be an biosynthetic intermediate. Thus, investigations of the existence of this putative NDP-octose intermediate and its transformation from octose 8-phosphate ( 8 ) have been the focus of our research effort.…”