Characterization of Early Enzymes Involved in TDP‐Aminodideoxypentose Biosynthesis en Route to Indolocarbazole AT2433

Abstract

The characterization of TDP-α-D-glucose dehydrogenase AtmS8, TDP-α-D-glucuronic acid decarboxylase AtmS9 and TDP-4-keto-α-D-xylose 2,3-dehydratase AtmS14 involved in Actinomadura melliaura AT2433 aminodideoxypentose is reported. This study provides the first biochemical evidence that both deoxypentose and deoxyhexose biosynthetic pathways share common strategies for sugar 2,3-dehydration/reduction and implicates the sugar nucleotide base specificity of AtmS14 as a potential mechanism for sugar nucleotide commitment to secondary metabolism. In addition, a re-evaluation of the AtmS9 homolog involved in calicheamicin aminodeoxpentose biosynthesis (CalS9) reveals CalS9 to catalyze UDP-4-keto-α-D-xylose as the predominant product rather than UDP-α-D-xylose as previously reported. Cumulatively, this work provides additional fundamental insights regarding the biosynthesis of novel pentoses attached to complex bacterial secondary metabolites.

EvdS6 is a bifunctional decarboxylase from the everninomicin gene cluster

“…However, with extended reaction timing and depletion of the NDP-GlcA substrate, NDP-4”-keto-D-xylose can re-enter the active site and drive the reaction to the formation of NDP-D-xylose. This was observed with ArnA, which was previously thought to only produce the keto product but extended incubation showed that ArnA will produce detectable levels of the reduced product, and conversely with CalS9, which was previously shown to only produce the reduced product but was later confirmed to release the oxidized NDP-4”-keto-pentose ( 39 , 43 ). The dissociation of the keto-product is required by the data herein, which showed that UDP-4”-keto-D-xylose is produced and released first but with extended (overnight) reaction UDP-D-xylose becomes the predominate product of EvdS6.…”

“…The conversion of UDP-glucuronic acid to UDP-xylose by a glucuronic acid decarboxylase was first confirmed in the fungus Cryptococcus laurentii ( 30 ). Since then this class of enzyme has also been characterized in a number of bacteria, including Escherichia coli , Streptomyces species, Ralstonia solanacearum , Sinorhizobium meliloti , Bacteroides fragilis , Rhodothermus marinus , and Actinomadura melliaura , and in eukaryotes including humans ( 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ). Glucuronic acid decarboxylases begin with NAD + -dependent oxidation at C-4” of NDP-GlcA, followed by decarboxylation of the C-6” carboxylate via an enolate intermediate, and then reduction of the C-4” position with NADH generated in the first phase of the reaction, thus regenerating the NAD + cofactor ( 30 ).…”

“…Glucuronic acid decarboxylases begin with NAD + -dependent oxidation at C-4” of NDP-GlcA, followed by decarboxylation of the C-6” carboxylate via an enolate intermediate, and then reduction of the C-4” position with NADH generated in the first phase of the reaction, thus regenerating the NAD + cofactor ( 30 ). With this reaction scheme, most glucuronic acid decarboxylases produce NDP-D-xylose ( 3 ); however, a few enzymes, ArnA, AtmS9, CalS9, and RsU4pxs, have been characterized to release the oxidized product NDP-4”-keto-D-xylose ( 2 ) ( 32 , 35 , 39 ). In the investigation of ArnA, an E. coli glucuronic acid decarboxylase, R619 was identified as having significant sequence identity across the enzyme subclass and was proposed to be the catalytic base that facilitates the decarboxylation reaction ( 31 ).…”

Structural Characterization of CalS8, a TDP-α-d-Glucose Dehydrogenase Involved in Calicheamicin Aminodideoxypentose Biosynthesis

“…Importantly, of the other putative TDGHs encoded by secondary metabolite gene clusters (Fig. 4B, At2433 AtmS8, esperamicin EspS8, and maduropeptin MdpA2), AtmS8 was also recently demonstrated to display a preference for TDP-Glc in vitro (63).…”

Chemistry and biology of specialized metabolites produced by Actinomadura

“…233 A cascade of genes involved in the biosynthesis of aminodideoxypentose was identied within the atm gene cluster, and the functions of several of these genes have been characterized. 233,243,244 Biosynthesis of aminodideoxypentose is initiated by the a-D-glucose-1-phosphate thymidyltransferase AtmS7, then the TDP-a-D-glucose dehydrogenase AtmS8, and the TDP-glucuronic acid decarboxylase AtmS9, catalyse C-6 oxidative decarboxylation. Both the TDP-4keto-a-D-xylose 2,3-dehydratase, AtmS14, and the TDP-2-deoxy-4-keto-a-D-pentos-2-ene 2,3-reductase, AtmS12, are required for C-2 deoxygenation.…”