Structure‐Guided Design of Formate Dehydrogenase for Regeneration of a Non‐Natural Redox Cofactor

Guo, Xiaojia; Liu, Yuxue; Li, Qing; Wang, Junting; Liu, Wujun; Zhao, Zongbao K.

doi:10.1002/chem.202003102

Cited by 20 publications

(8 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The herein studied FDH from Rhodobacter capsulatus ( Rc FDH) consists of a (αβγδ) 2 dimer of heterotetramers harboring a bis‐molybdopterin guanine dinucleotide (bis‐MGD) cofactor, in addition to five [4Fe‐4S] clusters, two [2Fe‐2S] clusters, and a flavin mononucleotide (FMN) prosthetic group [4] . Nicotinamide adenine dinucleotide (NAD + ) is the physiological electron acceptor [2a,4,5] . In its resting state, the Mo VI ion is coordinated by four sulfurs from two MGD dithiolenes, a sulfido ligand and a cysteine (Cys386) ligand from the protein backbone [2a,4] .…”

Section: Introductionmentioning

confidence: 99%

“… [4] Nicotinamide adenine dinucleotide (NAD + ) is the physiological electron acceptor. [ 2a , 4 , 5 ] In its resting state, the Mo VI ion is coordinated by four sulfurs from two MGD dithiolenes, a sulfido ligand and a cysteine (Cys386) ligand from the protein backbone. [ 2a , 4 ] Upon two electron reduction, the Mo IV ion is proposed to be pentacoordinated due to the displacement of Cys386.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Infrared Spectroscopy Elucidates the Inhibitor Binding Sites in a Metal‐Dependent Formate Dehydrogenase

Laun

Duffus

Wahlefeld

et al. 2022

Chemistry A European J

View full text Add to dashboard Cite

Biological carbon dioxide (CO 2 ) reduction is an important step by which organisms form valuable energyricher molecules required for further metabolic processes. The Mo-dependent formate dehydrogenase (FDH) from Rhodobacter capsulatus catalyzes reversible formate oxidation to CO 2 at a bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor. To elucidate potential substrate binding sites relevant for the mechanism, we studied herein the interaction with the inhibitory molecules azide and cyanate, which are isoelectronic to CO 2 and charged as formate. We employed infrared (IR) spectroscopy in combination with density functional theory (DFT) and inhibition kinetics. One distinct inhibitory molecule was found to bind to either a noncompetitive or a competitive binding site in the secondary coordination sphere of the active site. Site-directed mutagenesis of key amino acid residues in the vicinity of the bis-MGD cofactor revealed changes in both non-competitive and competitive binding, whereby the inhibitor is in case of the latter interaction presumably bound between the cofactor and the adjacent Arg587.

show abstract

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Infrared Spectroscopy Elucidates the Inhibitor Binding Sites in a Metal‐Dependent Formate Dehydrogenase

Laun

Duffus

Wahlefeld

et al. 2022

Chemistry A European J

View full text Add to dashboard Cite

show abstract

“…The mutant FalDH 9B2 gained high cofactor preference for NCD largely because of the formation of a shrunk ligand binding pocket. Further studies can be expected to use FalDH 9B2 in combination with previously developed NCD‐linked methanol dehydrogenase [17] and formate dehydrogenase [15b] to assemble orthogonal pathways for bioconversion of C1 molecules. Those pathways can then be explored for applications in synthetic and chemical biology studies.…”

Section: Discussionmentioning

confidence: 99%

Engineering Formaldehyde Dehydrogenase from Pseudomonas putida to Favor Nicotinamide Cytosine Dinucleotide

et al. 2022

Self Cite

View full text Add to dashboard Cite

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 dinucleo-tide (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.

show abstract

“…NCD is the only artificial coenzyme which was successfully biosynthesized and used in orthogonal redox reactions intracellularly, as demonstrated in our earlier studies [12][13][14] . Various NCD-favoring oxidoreductase, such as malic enzyme, phosphite dehydrogenase, d-lactate dehydrogenase, formate dehydrogenase, and formaldehyde dehydrogenase, have been successfully designed 2,[15][16][17][18] . However, these enzymes are not enough for the wide application of NCD in the future.…”

Section: Abbreviationsmentioning

confidence: 99%

A phosphite-based screening platform for identification of enzymes favoring nonnatural cofactors

Liu

Guo

et al. 2022

Sci Rep

Self Cite

View full text Add to dashboard Cite

Enzymes with dedicated cofactor preference are essential for advanced biocatalysis and biomanufacturing, especially when employing nonnatural nicotinamide cofactors in redox reactions. However, directed evolution of an enzyme to switch its cofactor preference is often hindered by the lack of efficient and affordable method for screening as the cofactor per se or the substrate can be prohibitively expensive. Here, we developed a growth-based selection platform to identify nonnatural cofactor-dependent oxidoreductase mutants. The growth of bacteria depended on the nicotinamide cytosine dinucleotide (NCD) mediated conversion of non-metabolizable phosphite into phosphate. The strain BW14329 lacking the ability to oxidize phosphite was suitable as host, and NCD-dependent phosphite dehydrogenase (Pdh*) is essential to the selection platform. Previously confirmed NCD synthetase with NCD synthesis capacity and NCD-dependent malic enzyme were successfully identified by using the platform. The feasibility of this strategy was successfully demonstrated using derived NCD-active malic enzyme as well as for the directed evolution of NCD synthetase in Escherichia coli. A phosphite-based screening platform was built for identification of enzymes favoring nonnatural cofactor NCD. In the future, once Pdh variants favoring other biomimetic or nonnatural cofactors are available this selection platform may be readily redesigned to attain new enzyme variants with anticipated cofactor preference, providing opportunities to further expand the chemical space of redox cofactors in chemical biology and synthetic biology.

show abstract

Structure‐Guided Design of Formate Dehydrogenase for Regeneration of a Non‐Natural Redox Cofactor

Cited by 20 publications

References 38 publications

Infrared Spectroscopy Elucidates the Inhibitor Binding Sites in a Metal‐Dependent Formate Dehydrogenase

Infrared Spectroscopy Elucidates the Inhibitor Binding Sites in a Metal‐Dependent Formate Dehydrogenase

Engineering Formaldehyde Dehydrogenase from Pseudomonas putida to Favor Nicotinamide Cytosine Dinucleotide

A phosphite-based screening platform for identification of enzymes favoring nonnatural cofactors

Contact Info

Product

Resources

About