Identification of cofactor discrimination sites in NAD-isocitrate dehydrogenase from Pyrococcus furiosus

Abstract

The role of Asp-328 and Ile-329 as a cofactor discrimination site of the NAD-dependent isocitrate dehydrognase (NAD-IDH) from Pyrococcus furiosus has been verified by replacing these residues with Lys and Tyr, respectively, which are the corresponding residues in NADP-IDH from Escherichia coli. The Asp-328-Lys mutant showed dual coenzyme specificity, whereas introduction of the double mutation, Asp-328-Lys/Ile-329-Tyr shifted the cofactor preference from NAD to NADP. NADP-dependent P. furiosus IDH retained thermostability and thermoactivity compared with NAD-IDH.

Biochemical Analysis of Halophilic Dehydrogenases Altered by Site-Directed Mutagenesis

“…The K cat /K m for the NADP + coenzyme was reduced 24-fold in the quadruple mutant. In contrast to the results obtained with other NAD(P)-dependent dehydrogenases (Nishiyama et al, 1993;Chen et al, 1997;Steen et al, 2002), our experiments showed that none of the mutants of Hfx. volcanii ICDH displayed dual cofactor specificity.…”

“…Both D343 (D278 in IMDH numbering) and I344 (I279 in IMDH numbering) are found in all known prokaryotic NAD-dependent decarboxylating dehydrogenases . In Pyrococcus furiosus NAD-dependent ICDH, the introduction of the double mutation of D328L/I329Y did not greatly decrease the preference for NAD + ; but it improved the preference for NADP + by reducing the catalytic efficiency for NAD + (Steen et al, 2002). In comparison, the loss of hydrogen bonding and the introduction of a negatively charged Asp greatly reduced binding with NADP + , but the changes had no effect on binding with NAD + in Hfx.…”

“…They are replaced with a variety of residues in the NADdependent ICDHs (Lloyd & Weitzman, 1988;Steen et al, 1997;Yasutake et al, 2003). Except for the substitution of Y391 with glutamine in Aeropyrum pernix, these residues are conserved in the archaeal NADP-dependent ICDHs and are in accordance with the cofactor specificity (Steen et al, 2002). However, K344 and Y345, which interact with NADP + in ICDH, are substituted by D278 and I279 in IMDH.…”

Isocitrate Dehydrogenase from the Hyperthermophile Aeropyrum pernix: X-ray Structure Analysis of a Ternary Enzyme–Substrate Complex and Thermal Stability

“…10,13,17 Only a few amino acid residues appear to be responsible for the discrimination between NAD C and NADP C . [18][19][20][21] The reaction mechanism of IDH has been extensively studied in EcIDH. 22,23 In the proposed mechanism, a proton is removed from the a-hydroxyl group of isocitrate.…”

Molecular Determinants of the Cofactor Specificity of Ribitol Dehydrogenase, a Short-Chain Dehydrogenase/Reductase

“…Altering the cofactor specificity of an enzyme in an artificial metabolic pathway can potentially correct the redox imbalance in the process and improve the overall product yield; therefore, cofactor engineering is important in applications ranging from cofactor regeneration to bioelectrocatalysis (3,4,12,17,19,21). A large body of literature describing the alteration of nicotinamide cofactor specificity is available (2,9,10,24,26,28,29,33,34), including the typical determinants and evolution of nicotinamide binding sites (7,8). Despite these attempts, alteration of cofactor specificity remains a challenge, since there are very few instances in which the catalytic efficiency of an initially disfavored cofactor has been suitably improved to match substrate specificity (5,13,29,34).…”