Structural Basis for 3-Amino-3-carboxypropyl Transfer in Nocardicin Biosynthesis

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• S-Adenosyl-l-methionine (SAM) is a versatile cofactor, acting as both a methyl donor and a 3-amino-3-carboxypropyl (3-ACP) donor.
• Nocardicin Amidotransferase (NAT) utilizes SAM to transfer the 3-ACP group to nocardicin G, producing isonocardicin C.
• The precise reaction mechanism of NAT has remained unclear due to limited structural data.

Purpose of the Study:

• To elucidate the structural basis of substrate recognition and the reaction mechanism of NAT.
• To provide a detailed understanding of 3-ACP transfer reactions catalyzed by NAT.

Main Methods:

• X-ray crystallography was employed to determine the apo and complex structures of NAT with nocardicin G and S-Adenosyl-homocysteine (SAH).
• Site-directed mutagenesis, thermal shift assay, molecular dynamics (MD) simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations were integrated for mechanistic analysis.

Main Results:

• The crystal structures revealed the key interactions involved in substrate binding and recognition by NAT.
• Mechanistic investigations demonstrated that the Cα-amino group of SAM acts as a Brønsted base, facilitated by E143, to activate the C6'-OH of nocardicin G.
• This activation promotes an SN2 attack on SAM, leading to the transfer of the 3-ACP group.

Conclusions:

• This study provides critical structural and computational insights into the mechanism of 3-ACP transfer by NAT.
• The findings clarify the role of SAM as a 3-ACP donor and highlight the catalytic strategies employed by NAT.
• This work advances the understanding of 3-ACP transferases and their biological significance.