Structural and Functional Basis of GenB2 Isomerase Activity from Gentamicin Biosynthesis

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Aminoglycosides, like gentamicin, are vital antibiotics against Gram-negative bacteria.
• Gentamicin's clinical use is limited by toxicity; its biosynthesis involves multiple enzymes.
• GenB2 is a pyridoxal-5'-phosphate (PLP)-dependent enzyme in gentamicin biosynthesis, responsible for epimerizing gentamicin C2 and C2a.

Purpose of the Study:

• To determine the structure of the GenB2 enzyme in its holo and ternary complexes.
• To elucidate the catalytic mechanism of GenB2, particularly its unique epimerization process.
• To understand the role of specific amino acid residues in GenB2's enzymatic activity.

Main Methods:

• X-ray crystallography was used to determine the structures of GenB2 in complex with PMP and substrate analogues.
• Site-directed mutagenesis was employed to investigate the function of key amino acid residues.
• Biochemical assays were performed to confirm the epimerase activity.

Main Results:

• The crystal structures of GenB2 revealed its holo and ternary complexes, providing insights into substrate binding.
• GenB2, a fold I PLP-dependent enzyme, utilizes a novel catalytic mechanism involving an N-terminal cysteine residue for epimerization.
• Mutagenesis of the N-terminal cysteine to serine or alanine abolished GenB2's epimerase activity, confirming its essential role.

Conclusions:

• This study reveals the structural basis and unique mechanism of GenB2, a novel fold I PLP-dependent epimerase.
• The findings enhance understanding of gentamicin biosynthesis enzymology.
• The research provides a foundation for engineering novel aminoglycoside derivatives through enzymatic synthesis.