Structural basis for the evolution of vancomycin resistance D,D-peptidases

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Vancomycin resistance in Gram-positive bacteria arises from altered cell-wall precursors (D-Ala-D-Lac or D-Ala-D-Ser) with low vancomycin binding affinity.
• The depletion of high-affinity precursors (D-Ala-D-Ala) is mediated by zinc-dependent D,D-peptidases like VanX and VanY.

Purpose of the Study:

• To elucidate the molecular basis for the diverse substrate specificities of Van D,D-peptidases, particularly the dual specificity of VanXY.
• To understand how these enzymes adapt to antibiotic pressure by targeting specific peptidoglycan precursors.

Main Methods:

• X-ray crystallography was used to determine the structures of VanXYC and VanXYG in apo, transition state analog-bound, and substrate/product-bound forms.
• Biochemical analyses were performed to investigate the catalytic mechanisms and substrate specificities.

Main Results:

• The crystal structures revealed that residues 110-115 in VanXY form a flexible mobile cap over the catalytic site, enabling hydrolysis of both dipeptides and pentapeptides.
• Structural comparisons showed that VanY enzymes lack this mobile cap, favoring pentapeptide hydrolysis over dipeptide hydrolysis.
• The study identified key structural determinants responsible for the selective hydrolysis of D-Ala-ending precursors over modified resistance targets.

Conclusions:

• The flexibility of the catalytic site cap is a key determinant of VanXY's dual specificity.
• Evolutionary adaptations in D,D-peptidases, driven by antibiotic pressure, have led to specific structural elements conferring hydrolytic activity against vancomycin-susceptible precursors.
• These findings provide insights into the adaptability of enzyme structures in response to antimicrobial agents.