Engineering catalytic properties and thermal stability of plant formate dehydrogenase by single-point mutations

Abstract

The analysis of the 3D model structure of the ternary complex of recombinant formate dehydrogenase from soya Glycine max (EC 1.2.1.2., SoyFDH) with bound NAD+ and an inhibitor azide ion revealed the presence of hydrophobic Phe290 in the coenzyme-binding domain. This residue should shield the enzyme active site from solvent. On the basis of the alignment of plant FDHs sequences, Asp, Asn and Ser were selected as candidates to substitute Phe290. Computer modeling indicated the formation of two (Ser and Asn) or three (Asp) new hydrogen bonds in such mutants. The mutant SoyFDHs were expressed in Escherichia coli, purified and characterized. All amino acid substitutions increased K(м)(HCOO-) from 1.5 to 4.1-5.0 mM, whereas the K(м)(NAD+) values remained almost unchanged in the range from 9.1 to 14.0 μM, which is close to wt-SoyFDH (13.3 μM). The catalytic constants for F290N, F290D and F290S mutants of SoyFDH equaled 2.8, 5.1 and 4.1 s⁻¹, respectively; while that of the wild-type enzyme was 2.9 s⁻¹. The thermal stability of all mutant SoyFDHs was much higher compared with the wild-type enzyme. The differential scanning calorimetry data were in agreement with the results of thermal inactivation kinetics. The mutations F290S, F290N and F290D introduced into SoyFDH increased the T(m) values by 2.9°C, 4.3°C and 7.8°C, respectively. The best mutant F290D exhibited thermal stability similar to that of FDH from the plant Arabidopsis thaliana and exceeded that of the enzymes from the yeast Candida boidinii and the bacterium Moraxella sp. C1.

The N-Terminal α-Helix Domain of Pseudomonas aeruginosa Lipoxygenase Is Required for Its Soluble Expression in Escherichia coli but Not for Catalysis

“…. In the enzyme structure, the covered hydrophobic regions and polar interactions are important to enzyme properties [2,5]. Thus, the exposed hydrophobic amino acids and the disrupted polar contacts and other potential noncovalent interactions created by removing the Nterminal domain may contribute to the decreased k c a t value, specific activity, and thermostability of the mini-LOX.…”

Study of the Structure-Function-Stability Relationships in Yeast D-amino Acid Oxidase: Hydrophobization of Alpha-Helices

“…In conclusion, we would like to mention that there are cases in protein engineering when a single amino acid substitution results in a significant stabilization of the enzyme [ 23 , 24 ]. However, usually the improvement of thermal stability can be achieved by combining several successful point mutations.…”

Современные Технологии Синтеза Фармацевтических Субстанций: На Пути К Высокоэффективному Производству

“…Ì. Â. Ëîìîíîñîâà [8 -11], èíñòèòóòà áèîõèìèè èì. À. Í. Áàõà [10,11], âíåäðåí÷åñêîé êîìïàíèè ÎÎÎ "Èííîâàöèè è âûñîêèå òåõíîëîãèè ÌÃÓ" [10,11], à òàêaeå êîëëåêòèâà àâòîðîâ êíèãè "Ôèçè÷åñêàÿ õèìèÿ áèîïðîöåññîâ" [12]. Îòìåòèì, â ÷àñòíîñòè, ãëàâû, ïîñâÿùåííûå ïîëó÷åíèþ ëþöèôåðàç ñ óëó÷øåííîé àêòèâíîñòüþ è ñòàáèëüíîñòüþ, â êîòîðûõ ïî-ëó÷èëè äàëüíåéøåå ðàçâèòèå ãåííî-èíaeåíåðíûå ìåòîäû ñîçäàíèÿ íîâûõ ôåðìåíòîâ.…”