Regioselective deacetylation based on teicoplanin-complexed Orf2* crystal structures.

Area of Science:

• Biochemistry
• Structural Biology
• Antibiotic Discovery

Background:

• Lipoglycopeptide antibiotics are more effective than vancomycin against MRSA.
• This enhanced efficacy is attributed to an additional aliphatic acyl side chain on glucosamine (Glm) at residue 4 (r4).
• The biosynthesis of the r4 N-acyl Glc moiety in teicoplanin (Tei) or A40926 involves Orf2* for precursor modification.

Purpose of the Study:

• To elucidate the structure of Orf2* in complex with substrates.
• To understand the molecular recognition mechanism of the r4 N-acyl Glc moiety.
• To engineer Orf2* for the synthesis of novel lipoglycopeptide analogs.

Main Methods:

• X-ray crystallography was used to solve two Orf2* structures.
• Structures were determined in complex with β-D-octyl glucoside and Tei.
• Site-directed mutagenesis was employed to create a triple-mutation mutant (S98A/V121A/F193Y).

Main Results:

• The crystal structures revealed the substrate binding site and a novel hydrophobic cavity within Orf2*.
• Molecular recognition of the r4 GlcNac moiety was identified as a key feature.
• The hydrophobic cavity accommodates substrates with longer acyl side chains.
• The triple-mutation mutant regioselectively deacetylated r6 GlcNac pseudo-Tei instead of r4 GlcNac.

Conclusions:

• The structural and biochemical data provide mechanistic insights into Orf2* function.
• The engineered Orf2* mutant enables regioselective modification of the r6 sugar moiety.
• This opens avenues for creating novel lipoglycopeptide analogs with potentially improved therapeutic properties.