Directed Evolution and Structural Analysis of NADPH-Dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) Reductase from Ralstonia eutropha Reveals Two Mutations Responsible for Enhanced Kinetics

Abstract

NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with ␤-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited k cat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.

Anaerobic poly-3-d-hydroxybutyrate production from xylose in recombinant Saccharomyces cerevisiae using a NADH-dependent acetoacetyl-CoA reductase

“…Since the crystal structures of these AARs were not available, protein structures were made with automated homology modeling using SWISS-MODEL [22]. The AvAAR displayed 56% protein sequence identity to the C. necator AAR whose crystal structure was used as template (PDB: 3VZP) [23]. The S. wolfei AARs had a protein sequence identity of 30% (GenBank ABI67978.1) and 36% (GenBank ABI69207.1) respectively, compared to the CnAAR.…”

“…The S. wolfei AARs had a protein sequence identity of 30% (GenBank ABI67978.1) and 36% (GenBank ABI69207.1) respectively, compared to the CnAAR. Both S. wolfei homology models ( Swol_0651 modelled on PDB: 4MOW [27] and Swol_1910 modelled on PDB: 4DMM [28]) indicated a NADPH-binding arginine motif [23], typical of the CnAAR and related AARs. In contrast, the cofactor binding site of the AvAAR contained a dominant glutamate instead of an arginine (compared to the C. necator AAR) (Fig.…”

“…1). Acidic residues, such as glutamate in the case of the AvAAR, can create hydrogen bonds to the 2′- and 3′-hydroxyl groups on the adenine ribose of NADH [29], in comparison to arginine, which are ideally forming contacts to the 2′-phosphate in NADPH [23]. The AvAAR was also assumed to generate the correct enantiomer, ( R )-3-hydroxybutyryl-CoA, preferred by the C. necator polyhydroxyalkanoate synthase for PHB accumulation as the binding site for the prochiral keto functionality on the acetoacetyl-CoA substrate was almost identical in the AvAAR homology model as in the CnAAR crystal structure.…”

“…The translated AAR gene from A. vinosum (GenBank Accession No. YP_003442070.1) was submitted, using the C. necator AAR structure as designated template (PDB: 3VZP) [23]. …”

Biosynthesis of Trehangelin in Polymorphospora rubra K07–0510: Identification of Metabolic Pathway to Angelyl‐CoA

“…ThgK is related to the classical SDR family enzyme β‐ketoacyl‐ACP reductase (BKR), which is involved in fatty acid biosynthesis by 3‐oxoacyl‐ACP reduction to form 3‐hydroxy‐ACP. Although NADPH‐dependent and NADH (nicotinamide adenine dinucleotide)‐dependent BKRs have been reported, ThgK required NADPH for the catalytic reaction. Thus it was concluded that ThgK is an NADPH‐dependent reductase that catalyses the formation of HMB‐CoA from MAA‐CoA.…”