Site Saturation Mutagenesis Applications onCandida methylicaFormate Dehydrogenase

Abstract

In NADH regeneration, Candida methylica formate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependent cmFDH from NAD+ to NADP+ and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+ to 5.6 × 104-fold and 5 × 104-fold value, respectively. To increase the thermostability of cmFDH, the conserved residue at position 1 in the catalytic domain of cmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating “smarter libraries” for improving the properties of cmFDH.

Involvement of SlSTOP1 regulated SlFDH expression in aluminum tolerance by reducing NAD+ to NADH in the tomato root apex

“…In this assay, NBT receives electrons from NADH via PMS and generates blue‐purple formazan. Substrate specificity was analyzed by observing the color change based on the reduction of NBT to soluble blue‐purple formazan in the presence of PMS, which reacts with the NAD(P)H produced by dehydrogenases (Debnam & Shearer, 1997; Özgün et al., 2016), with the various substrates citric acid, succinic acid, dl ‐malic acid, sodium formate and sodium oxalate at a concentration of 50 m m . NaCl was used as a control.…”

Stairway to Stereoisomers: Engineering Short‐ and Medium‐Chain Ketoreductases To Produce Chiral Alcohols

“…For example, The Candida methylica formate dehydrogenase (cmFDH) is an NADH generating enzyme. It is mutated (D195S/Q197T and D195S/Y196L) through SSM to use NADP + in vivo as SDRs predominantly use NADPH, a stoichiometrically lesser cofactor than NADH [52] . Based on the availability of infrastructure, this concept is extended to make a library of proteins where every amino acid is substituted with other 19 residues leading to large libraries (20× protein size).…”

“…It is mutated (D195S/Q197T and D195S/Y196L) through SSM to use NADP + in vivo as SDRs predominantly use NADPH, a stoichiometrically lesser cofactor than NADH. [52] Based on the availability of infrastructure, this concept is extended to make a library of proteins where every amino acid is substituted with other 19 residues leading to large libraries (20 × protein size). This probability is significantly reduced by using iterative saturation mutagenesis (ISM).…”

Switching the Cofactor Preference of Formate Dehydrogenase to Develop an NADPH-Dependent Biocatalytic System for Synthesizing Chiral Amino Acids

“…The FDH-based cofactor regeneration system for NADH cofactor regeneration is successful because most native FDHs efficiently transfer reducing power from formate to NAD + . However, several redox biocatalytic systems rely on NADPH rather than NADH. , The main limitation of the FDH-based NADPH regeneration system is its low catalytic performance ( k cat / K m of most native FDHs <10 mM –1 s –1 ), − low substrate affinity ( K m > 10 mM), , and poor specificity toward NADP + . The ratio between the catalytic efficiency of FDHs for NADP + and NAD + substrates, expressed as ( k cat / K m ) NADP+ /( k cat / K m ) NAD+ , is typically below 30.…”