Abstract
4-Hydroxyphenylacetate 3-hydroxylase (EcHpaB) from Escherichia coli is capable of efficient ortho-hydroxylation of a wide range of phenolic compounds and demonstrates great potential for broad chemoenzymatic applications. To understand the structural and mechanistic basis of its catalytic versatility, we elucidated the crystal structure of EcHpaB by X-ray crystallography, which revealed a unique loop structure covering the active site. We further performed mutagenesis studies of this loop to probe its role in substrate specificity and catalytic activity. Our results not only showed the loop has great plasticity and strong tolerance towards extensive mutagenesis, but also suggested a flexible loop that enables the entrance and stable binding of substrates into the active site is the key factor to the enzyme catalytic versatility. These findings lay the groundwork for editing the loop sequence and structure for generation of EcHpaB mutants with improved performance for broader laboratory and industrial use.
Highlights
FAD as prosthetic group to hydroxylate their substrates, which provide faster substrate turnover than CYP450 enzymes[14,15]
The highest titers were reported at 62.7 mg/L and 34.7 mg/L in whole-cell catalysis studies, respectively[18]. These results indicate that EcHpaB has a broad substrate spectrum for the hydroxylation of a variety of phenolic compounds
The further sequence and structural analysis suggested that the enlarged entrance of substrate-binding pocket and improved flexibility of the loop structure caused by the changes in the amino acid sequence and the corresponding changes in the loop structure may play important roles in the improved catalytic versatility of the mutant
Summary
FAD as prosthetic group to hydroxylate their substrates, which provide faster substrate turnover than CYP450 enzymes[14,15]. We further used a set of natural and non-natural substrates with gradually increased molecular sizes to survey the catalytic properties changes among these mutants through in vivo and in vitro enzymes assays These efforts led to the identification of a mutant (XS6) demonstrating increased activity towards non-natural larger substrates resveratrol and naringenin. The further sequence and structural analysis suggested that the enlarged entrance of substrate-binding pocket and improved flexibility of the loop structure caused by the changes in the amino acid sequence and the corresponding changes in the loop structure may play important roles in the improved catalytic versatility of the mutant Overall, these studies provide structural insights into the substrate preference of this FAD-dependent hydroxylase and suggest a key sequence and structural feature for protein engineering efforts aiming at altering the catalytic properties and versatilities of these type of enzymes for broader laboratory and industrial use
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