Abstract
The emergence of multidrug-resistant Gram-positive pathogens is a serious public health issue. Thus, the development of novel glycopeptide antibiotics with higher efficacy against resistant strains is urgently demanded. Lipoglycopeptide antibiotics, such as teicoplanin (Tei) and A40926, are more effective than vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) in as much as they carry an extra aliphatic acyl side chain on glucosamine (Glm) at residue 4 (r4). In this pursuit, we attempted to reposition N-acyl Glc from r4 to r6 at Tei by structure- and function-based protein engineering. The biosynthesis of the r4 N-acyl Glc moiety at Tei or A40926 has been elucidated, in which the primary amine nucleophile of Glm is freed from the r4 GlcNac pseudo-Tei precursor by Orf2* for the subsequent acylation reaction to occur. In this dissertation, two Orf2* structures in complex with β-d-octyl glucoside or Tei were solved. Of the complexed structures, the substrate binding site and a previously unknown hydrophobic cavity were revealed, wherein r4 GlcNac acts as the key signature for molecular recognition and the cavity allows substrates carrying longer acyl side chains in addition to the acetyl group. On the basis of the complexed structures, a triple-mutation mutant S98A/V121A/F193Y is able to regioselectively deacetylate r6 GlcNac pseudo-Tei instead of that at r4. On the other hand, a methyltransferase Dbv27 from A40926 biosynthesis pathway was found to be able to double methylate r1 free amine group so that Dbv27 can be used in protection chemistry. Thereby, novel analogs can be selectively made at the r6 sugar moiety through simple organic synthesis. On the other hand, we are interested in the biosynthesis of nonproteinogenic amino acids because such study is informative in developing new antibiotics. In this study, we explored the biosynthesis of 刍-hydroxy-enduracididine in mannopeptimycin. Four gene products (MppOPQR) were predicted to be involved in the synthesis. The gene products of mppPQ were predicted to be PLP-dependent aminotransferases, while mppR has no functionally known homolog. MppO recently was characterized to be able to hydroxylate enduracididine at the 刍-carbon position. To understand how enduracididine is synthesized, we focus on MppP, MppQ and MppR. MppP and MppQ proteins unfortunately were found to be insoluble in E. coli. Nevertheless, the crystal structures of MppR and the mutant K156A have now been solved. MppR forms as tetramers in solution. Comparing K156A with native MppR, there is no obvious deviation from corresponding residues within the putative active site. In MALDI-MS analysis, the protein fragmentations between the native and K156A MppR suggested that there is an extra moiety covalently bound to Lys156. On the basis of the contour in the density map, this moiety seems to be the precursor of enduracididine. Although the real function of MppR in the biosynthesis of enduracididine remains unknown, this study however has paved a solid foundation for further study in this regard.