Graduate Thesis Or Dissertation
 

Studies on the ecological functions of the antidiabetic drugs acarbose

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  • Natural products (a.k.a. secondary metabolites) are produced by living organisms that are not essential for their growth, development, or reproduction, but may play a role in their survival or adaptability in nature. Many secondary metabolites isolated from plants and microbes have found use as molecular probes, drug leads, or medicine to treat diseases in humans, animals, or plants. However, their benefit to the producing organisms is rarely understood. Among secondary metabolites that are used to treat human diseases is the antidiabetic drug acarbose. This pseudo-oligosaccharide has strong inhibitory activity towards a wide variety of glycosyl hydrolases (GH). Although many bacteria (particularly actinobacteria) produce acarbose or related compounds, their ecological or biological roles in nature have not been fully understood. Currently, there are two hypotheses related to the ecological functions of acarbose, the competitive model and the carbophore model. The competitive model refers to acarbose being used as a tool to compete with other organisms for food by inhibiting their sugar hydrolase enzymes, whereas the carbophore model suggests that acarbose is used as a sugar transporter, shuttling in and out between intra- and extracellular spaces of bacteria and transporting glucose (G) and/or maltose (G2) from outside to inside the cell. However, there was almost no experimental data to support either one of these hypotheses. To investigate the ecological and/or biological role of acarbose, we tested the possibility of the competitive model as well as the carbophore model by in vitro and in vivo experiments. First, we examined the competitive model by culturing acarbose producing actinomycetes and a non-producing bacterium (Streptomyces coelicolor) in a minimum medium where starch was the only carbon source. We found that in the absent of acarbose, all tested bacteria were able to grow, but in the presence of acarbose, only the acarbose-producing bacteria were able to grow normally, suggesting that these bacteria produce GH enzymes that are resistant to acarbose. Indeed, a number of GH genes have been identified within the acarbose biosynthetic gene clusters (BGC). We cloned several of these genes, produced the recombinant proteins in Escherichia coli, and characterized their function and sensitivity to acarbose. The results showed that all acarbose-producing actinomycetes are equipped with at least one acarbose-resistant α-amylase, whose genes are located within the acarbose BGC. On the other hand, a comparable α-amylase from S. coelicolor was found to be sensitive to acarbose. Multiple amino acid sequence alignment of acarbose-resistant and acarbose-sensitive α- amylases revealed a single amino acid residue (His-304) that is mostly responsible for the acarbose-sensitive phenotype of the enzymes. Protein modeling studies showed that α-amylases with His-304 have a smaller active site pocket compared to those that contain asparagine or alanine at the same position. The acarbose-resistant amylase AcbE from Actinoplanes sp. SE50/110 contains alanine at this position. Point mutation of A304H in AcbE resulted in an acarbose-sensitive AcbE variant. Together, the data support the notion that acarbose is used by the producing bacteria to compete with other organisms for sugars. Using acarbose-resistant amylases as query we further identified many more acarbose or other pseudo-oligosaccharide biosynthetic gene clusters in over 100 strains of actinomycetes, suggesting that this class of natural products provide a significant benefit to the producing organisms in their natural environment. As for the carbophore model, we investigated two other sugar processing enzymes, AcbD and AcbQ, which are encoded in the acarbose BGC. The extracellular transglycosidase (TG) enzyme AcbD has been shown previously to transfer glucose (G) and maltose (G2) from oligosaccharides to acarbose to form acarbose-G and acarbose-G2, respectively. It is hypothesized that the extended acarbose derivatives are transported into the cell and phosphorylated by the putative acarbose kinase, AcbK. The phosphorylated acarbose-G (acarbose-G 7-phosphate) and acarbose-G2 7-phosphate are then hydrolyzed by the putative GH AcbQ, releasing glucose to the cytosol for cell growth and metabolism. To test this hypothesis, we produced recombinant AcbD and AcbQ and characterized their GH and TG activities. Contrary to what originally proposed, we found that AcbQ is more a TG than a GH. Although AcbQ can hydrolyze acarbose-G (or acarbose-G2) to produce acarbose and glucose, the catalytic efficiency is low. In contrast, AcbQ can effectively catalyze TG reactions between acarbose and G2 or maltotriose (G3) to give acarbose-G or acarbose-G2, respectively. Each of these reactions produces glucose. The enzyme can also catalyze TG reaction of acarbose-G1 or acarbose 7-phosphate with G2 or G3. Each of these reactions also produce glucose, suggesting that the primary function of AcbQ is to hydrolyze G2 and G3 to glucose via TG reactions. It has been reported that the acarbose producing strain Actinoplanes sp. SE50/110 can express an active transporter for the intake of G2 and G3. However, how G2 and G3 are utilized in the cell is still an open question. We propose that the intracellular TG reactions of acarbose and its derivatives with G2 and G3 by AcbQ to give glucose as the net product is another benefit of acarbose for the producing bacteria. In summary, our study suggests that acarbose is produced by bacteria to compete with other bacteria for sugars by inhibiting their α-amylase enzymes. The compound is also used as a tool to produce more glucose in the cell by breaking down G2 and G3 through TG reactions involving AcbQ as a catalyst.
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