Regioselective Control of SAM Halogenation through Structure-Guided Directed Evolution of a Hydroxide...

Area of Science:

• Biocatalysis and Enzyme Engineering
• Synthetic Biology
• Biochemistry

Background:

• Enzymatic halogenation offers mild reaction conditions, contrasting with traditional chemical methods.
• S-adenosylmethionine (SAM) is a key cofactor for SAM-dependent halogenases in antibiotic biosynthesis.
• Hydroxide adenosyltransferases (HATases) and SAM-dependent halogenases share the DUF-62 protein family, but HATases use water, not SAM.

Purpose of the Study:

• To engineer a HATase for regioselective control of SAM halogenation, a feat not previously achieved.
• To identify key protein residues and mutations that dictate regioselectivity in SAM cleavage.
• To explore the potential of the DUF-62 superfamily for regioselective SAM modification.

Main Methods:

• Protein engineering of a HATase from *Thermotoga maritima* MSB8.
• Directed evolution using *E. coli* cell surface display to generate functional mutants.
• Computational studies to analyze mutation effects on halide orientation and SAM cleavage regioselectivity.

Main Results:

• Engineered HATase mutants successfully converted SAM to methyl iodide (CH3I) and S-adenosylhomocysteine (SAH).
• Identified key residues that influence the regioselective cleavage of SAM.
• These mutations conferred regioselective halogenation capabilities to other HATases, demonstrating broad applicability.

Conclusions:

• The study demonstrates successful engineering of HATases for regioselective SAM cleavage, creating novel halide methyltransferases.
• This work highlights the DUF-62 protein family's potential for precise SAM modification.
• The engineered enzymes expand the toolkit for biocatalytic halogenation and chemical synthesis.