Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity

Abstract

Changing the cofactor specificity of an enzyme from nicotinamide adenine dinucleotide 2′-phosphate (NADPH) to the more abundant NADH is a common strategy for increasing overall enzyme efficiency in microbial metabolic engineering. The aim of this study was to switch the cofactor specificity of the primary–secondary alcohol dehydrogenase from Clostridium autoethanogenum, a bacterium with considerable promise for the bio-manufacturing of fuels and other petrochemicals, from strictly NADPH-dependent to NADH-dependent. We used insights from a homology model to build a site-saturation library focussed on residue S199, the position deemed most likely to disrupt binding of the 2′-phosphate of NADPH. Although the CaADH(S199X) library did not yield any NADH-dependent enzymes, it did reveal that substitutions at the cofactor phosphate-binding site can cause unanticipated changes in the substrate specificity of the enzyme. Using consensus-guided site-directed mutagenesis, we were able to create an enzyme that was stringently NADH-dependent, albeit with a concomitant reduction in activity. This study highlights the role that distal residues play in substrate specificity and the complexity of enzyme–cofactor interactions.

Crystal structures of human 17β‐hydroxysteroid dehydrogenase type 1 complexed with estrone and NADP+ reveal the mechanism of substrate inhibition

“…Mutagenesis study demonstrated that the sole mutation of the Leu 36 into aspartic acid residue indeed changed the cofactor preference of 17β‐HSD1 from NADP(H) to NAD(H) , and eliminated the substrate inhibition of the enzyme in the presence of NADPH . Although the (phosphor‐)adenosine moiety of NADP is distal from the catalytic site of the enzyme, subtle perturbations to the (phosphor‐) adenosine binding pocket was proved to have a dramatic effect on activity and mutations at the 2′‐phosphate binding site was demonstrated to affect substrate specificity . Thus the stabilization of the 2′‐phosphate group of NADPH is essential for maintaining the substrate inhibition in 17β‐HSD1.…”

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

“…However, in many cases the mutations do not achieve the desired effect, cause the loss of catalytic activity or lead to unexpected results [185,186]. This is partially due to the lack of understanding of the relationship between the amino acid sequence of a protein, its structure and its function, which still prevents us from developing widely applicable methodologies to alter the substrate or cofactor specificity of oxidoreductases.…”

A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases

“…-1.1 (Kristan et al 2007) C. lunatus BHSDH 3QWF Y49D 7.8 ? -4.2 (Liang et al 2007) P. stipitis XR 1K8C (h) K270R, N272D 2.9 9.3 -1.2 (Maddock et al 2015) E. coli CaADH 1KEV (h) G198D, S199V, P201E, Y218A >1 ? -4.6 (Maurer et al 2005) B Log Relative Activity c (Medina et al 2001) A. PCC7119 FDNR 2BSA S223D 0.12 8.1x10 3 -5.4 (Nakanishi et al 1997) M. musculus CR 1CYD T38D 31 1.3x10 3 -0.51 (Paladini et al 2009 (Schepens et al 2000) S. bicolor MDH 7MDH (1CIV) G84D, S85I, R87Q, S88A 12 2.1x10 4 -0.96 (Scrutton et al 1990) E. coli GTR 1GET A179G, A183G, V197E, R198M, K199F, H200D, R204P 8.1 1.8x10 4 -1.5 (Shiraishi et al 1998) N. crassa CbR S920D, R932S 65 7.2x10 4 -2.6 (Takase et al 2014) S. sp.…”