Modular exchange of substrate-binding loops alters both substrate and cofactor specificity in a member of the aldo-keto reductase superfamily

Abstract

Substrate specificity in the aldo-keto reductase (AKR) superfamily is determined by three mobile loops positioned at the top of the canonical (α/β)(8)-barrel structure. These loops have previously been demonstrated to be modular in a well-studied class of AKRs, in that exchanging loops between two similar hydroxysteroid dehydrogenases resulted in a complete alteration of substrate specificity (Ma,H. and Penning,T.M. (1999) Proc. Natl Acad. Sci. USA, 96, 11161-11166). Here, we further examine the modularity of these loops by grafting those from human aldose reductase (hAR) into the hyperthermostable AKR, alcohol dehydrogenase D (AdhD), from Pyrococcus furiosus. Replacement of Loops A and B was sufficient to impart hAR activity into AdhD, and the resulting chimera retained the thermostability of the parent enzyme. However, no active chimeras were observed when the hAR loops were grafted into a previously engineered cofactor specificity mutant of AdhD, which displayed similar kinetics to hAR with the model substrate dl-glyceraldehyde. The non-additivity of these mutations suggests that efficient turnover is more dependent on the relative positioning of the cofactor and substrate in the active site than on binding of the individual species. The ability to impart the substrate specificities of mesostable AKRs into a thermostable scaffold will be useful in a variety of applications including immobilized enzyme systems for bioelectrocatalysis and fine chemical synthesis.

Highly Diverse Protein Library Based on the Ubiquitous (β/α)8 Enzyme Fold Yields Well‐Structured Proteins through in Vitro Folding Selection

“…The GDPD catalytic face consists mainly of short loops and could potentially accommodate larger active sites with minimal steric clashes, similar to recent experiments that changed TIM barrel activities. [7c] To optimize our protocols for folding assessment and selection that are essential to our library assembly strategy, we prepared a destabilized GDPD construct (GDPDmut) lacking the parental tertiary structure. In particular, two adjacent substitutions (G31R/V32E) were introduced to the (β/α) 8 barrel to disrupt the parent GDPD structure (GDPDwt) via steric clashing and the insertion of unfavorable charge in the tightly packed core of the barrel.…”

“…[1b, 1c, 6] In an effort to enable more divergent sequence exploration well beyond that obtainable by point mutations, the tolerance of (β/α) 8 scaffolds to the insertion of different natural (β/α) 8 loop fragments was investigated. [7] Furthermore, the enzymatic activity of existing (β/α) 8 barrel proteins was improved or modified by a combination of rational design and directed evolution similar to proteins of other folds. [2c, 8] In addition, rational design approaches for de novo enzymes repeatedly favored the (β/α) 8 barrel fold over others, likely due to its ability to appropriately position catalytic and substrate binding residues.…”

Robust enzyme design: Bioinformatic tools for improved protein stability

“…4). For example, loops within the (α/β) 8 -barrel structure that determine substrate binding and specificity of a mesostable human aldose reductase were introduced into its thermostable homolog -alcohol dehydrogenase D from Pyrococcus furiosus [78]. The resulting chimera had improved substrate specificity and retained the high temperature tolerance of the parent enzyme.…”

Acceleration of an aldo-keto reductase by minimal loop engineering

“…However, mobile loops also slow down the reaction rate ( k cat of max ∼30 s −1 ; e.g. Ma and Penning, 1999; Couture et al , 2004; Kratzer et al , 2006; Campbell et al , 2013) and add complexity to structure–function relationships (Couture et al , 2004), thereby impairing AKR application in biocatalysis. A previous study aimed at inverting the substrate specificities of two mammalian hydroxysteroid dehydrogenases that act on opposite ends of steroid hormone substrates.…”

“…Point mutations of substrate-binding residues were not sufficient to reverse substrate specificities despite the close relatedness of the enzymes (67% sequence identity) (Ma and Penning, 1999). Recently, grafting of loops from human aldose reductase into a hyperthermostable alcohol dehydrogenase gave a hint that substrate and coenzyme binding by AKRs is not entirely independent (Campbell et al , 2013). In the present study, we optimized an AKR for the reduction of bulky-bulky ketones by minimal loop engineering.…”