Mining and engineering of valine dehydrogenases from a hot spring sediment metagenome for the synthesis of chiral non-natural L-amino acids

Valine dehydrogenases (ValDHs) represent promising candidates for the development of biocatalysts for short chain amino acids or amines synthesis. However, only a limited number of ValDHs have been reported. Metagenomic mining is a powerful tool to access new members of limited enzyme families. Here we report the discovery of two novel ValDHs (HsValDH3 and HsValDH4) by metagenomic mining of a hot spring sediment sample. After systematical characterization and molecular dynamics simulations of HsValDH3 and HsValDH4, an interesting correlation between their structure compactness and thermostability was uncovered. We also identified a residue (I294) in the active pocket of HsValDH3 that plays a crucial role in its substrate specificity. Site-directed mutagenesis of this residue successfully shifted the substrate preference of HsValDH3, revealing a possible evolutionary route between ValDHs and LeuDHs. HsValDH3 and its mutant showed excellent catalytic efficiency and stereoselectivity in the synthesis of three valuable non-natural L-amino acids, highlighting their potential practical applications.