4.4 Article

Engineering Formaldehyde Dehydrogenase from Pseudomonas putida to Favor Nicotinamide Cytosine Dinucleotide

期刊

CHEMBIOCHEM
卷 23, 期 7, 页码 -

出版社

WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.202100697

关键词

binding pocket; cofactor preference; formaldehyde dehydrogenases; nicotinamide cytosine dinucleotides; non-natural cofactors

资金

  1. National Key R&D Program of China [2019YFA0904900]
  2. National Natural Science Foundation of China [21837002, 21877112, 21721004]
  3. Dalian Institute of Chemical Physics, CAS [DICP Bio-ChE-X201801]
  4. China Postdoctoral Science Fund [2020M670805]

向作者/读者索取更多资源

This study demonstrates that FalDH variants can be designed to prefer non-natural cofactor NCD by structure-guided modification of the binding pocket. The best variant, FalDH 9B2, showed over 150-fold higher preference for NCD compared to NAD. These engineered enzymes, along with other NCD-linked enzymes, provide opportunities for the biological conversion of C1 molecules.
The enzyme formaldehyde dehydrogenase (FalDH) from Pseudomonas putida is of particular interest for biotechnological applications as it catalyzes the oxidation of formaldehyde independent of glutathione. However, the consumption of a stoichiometric amount of nicotinamide adenine dinucleotide (NAD) can be challenging at the metabolic level as this may affect many other NAD-linked processes. A potential solution is to engineer FalDH to utilize non-natural cofactors. Here we devised FalDH variants to favor nicotinamide cytosine dinucleotide (NCD) by structure-guided modification of the binding pocket for the adenine moiety of NAD. Several mutants were obtained and the best one FalDH 9B2 had over 150-fold higher preference for NCD than NAD. Molecular docking analysis indicated that the cofactor binding pocket shrunk to better fit NCD, a smaller-sized cofactor. FalDH 9B2 together with other NCD-linked enzymes offer opportunities to assemble orthogonal pathways for biological conversion of C1 molecules.

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