Molecular Insights into Converting Hydroxide Adenosyltransferase into Halogenase

Area of Science:

• Biochemistry
• Enzymology
• Synthetic Biology

Background:

• Enzymatic halogenation is crucial for agrochemical development, offering specificity and mild conditions.
• Existing S-adenosyl-l-methionine (SAM)-dependent halogenases are limited, with few known fluorinases and chlorinases.
• SAM-dependent hydroxide adenosyltransferases (HATases) share reaction mechanisms with halogenases but use water instead of halides.

Purpose of the Study:

• To engineer a novel halogenase from a thermophilic HATase.
• To identify key mutations conferring halogenation activity and halide specificity.
• To develop thermostable halogenases for industrial applications.

Main Methods:

• Exploration of a HATase from *Thermotoga maritima* MSB8.
• Site-directed mutagenesis to identify key residues (W8L/V71T dyad).
• Characterization of mutant enzymes (M1, M2, M4) for Cl-, Br-, and I- halogenation.
• Quantum mechanics/molecular mechanics (QM/MM) simulations to elucidate binding mechanisms.

Main Results:

• Successful transformation of HATase into a functional halogenase.
• Identification of a key W8L/V71T dyad for halogenation.
• Development of high-performing mutants (M1, M2, M4) for specific halide incorporation.
• M4 mutant demonstrated thermostability at 80 °C for iodination, outperforming natural halogenases.
• QM/MM revealed optimized halide binding angles for SAM nucleophilic attack.

Conclusions:

• Engineered HATase exhibits halide ion specificity, creating novel halogenases.
• Generated the first thermostable halogenases, expanding the enzyme repertoire.
• Provides new avenues for agrochemical design and enzymatic synthesis of halogenated compounds.