| Summary: | 碩士 === 國立臺灣海洋大學 === 食品科學系 === 95 === Vancomycin and teicoplanin that are the only two currently licensed glycopeptide antibiotics act as the last resort treatment against serious Gram positive bacterial infections, such as methicillin-resistant Staphylococcus aureus (MRSA). With the emergence of vancomycin-resistant enterococci (VRE) and the more serious vancomycin-resistant Staphylococcus aureus (VRSA), the development of novel glycopeptide antibiotics with higher efficacy against resistant strains is therefore highly demanded. The approach through genetic manipulation of secondary metabolite biosynthetic pathways has been shown promising and indeed become a hot research area. To achieve this, the biological function of each gene product involved in the biosynthesis for a particular metabolite however needs to be known at the first place. Here we study the functions of three homologous (> 65% identity) proteins Dbv21, Orf2*, and Orf15, previously assigned as hypothetical proteins in the biosynthesis of A40926, teicoplanin, and chloroeremomycin, respectively. The results indicate that Dbv21 and Orf2* are deacetylases and Orf15 is a thymidyltransferase. In addition, the deacetylation of Dbv21 and Orf2* is occurring at the stage of N-acetyl glucosaminyl pseudoaglycone. On the other hand, the results also revealed that dTTP should be the physiological sugar activator for Orf15 with a co-substrate either glucose-1-phosphate or glucosamine-1-phosphate or N-acetyl-glucosamine-1-phosphate together to produce a molecule of corresponding dTDP-sugar.
It is extremely interesting to learn that the two homologues have 65% protein sequence identity (72% similarity) but play completely different roles, perhaps only by subtle changes in their active sites. Sequence and structural alignments in particular with the MshB structure suggested that a number of highly conserved residues may play pivotal roles toward the diversity. In particular, there are two regions essential for catalytic activity that are conserved throughout all homologues: they are 15-19, (A/P)H(P/L/A)DD, and 161-164, HXD(H/N), corresponding to Dbv21 residues. We therefore examined the activities of the deacylation of teicoplanin as well as the formation of dTDP-glucose over the selected mutants (Dbv21-D18N/T, -D19V and -H161F and Orf15-D18V and -H16F). The biochemical results revealed that Asp18 and His161 of Dbv21 as well as Asp18 and His16 of Orf15 are all critical and may all coordinate with a metal ion per se. Evidence that were provided herein should lead us an insight into the catalytic mechanisms for these previous unknown proteins. Detailed structure-function analyses of these enzymes should also expand our knowledge in the two important classes of enzymes − deacetylases and thymidyltransferases.
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