Engineering of Ancestors as a Tool to Elucidate Structure, Mechanism, and Specificity of Extant Terpene Cyclase

Abstract

Structural information is crucial for understanding catalytic mechanisms and to guide enzyme engineering efforts of biocatalysts, such as terpene cyclases. However, low sequence similarity can impede homology modeling, and inherent protein instability presents challenges for structural studies. We hypothesized that X-ray crystallography of engineered thermostable ancestral enzymes can enable access to reliable homology models of extant biocatalysts. We have applied this concept in concert with molecular modeling and enzymatic assays to understand the structure activity relationship of spiroviolene synthase, a class I terpene cyclase, aiming to engineer its specificity. Engineering a surface patch in the reconstructed ancestor afforded a template structure for generation of a high-confidence homology model of the extant enzyme. On the basis of structural considerations, we designed and crystallized ancestral variants with single residue exchanges that exhibited tailored substrate specificity and preserved thermostability. We show how the two single amino acid alterations identified in the ancestral scaffold can be transferred to the extant enzyme, conferring a specificity switch that impacts the extant enzyme’s specificity for formation of the diterpene spiroviolene over formation of sesquiterpenes hedycaryol and farnesol by up to 25-fold. This study emphasizes the value of ancestral sequence reconstruction combined with enzyme engineering as a versatile tool in chemical biology.

Combining ancestral reconstruction with folding-landscape simulations to engineer heterologous protein expression

“…4d) and, according to our working hypothesis, we do not expect its replacement with the corresponding ancestral sequence to improve the efficiency of heterologous folding. The experimental results on CPk- [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] thioredoxin conform to this expectation (Fig. 5c and d).…”

“…We followed procedures we have previously described in several publications [47][48][49]60 with small modifications. Briefly, genes encoding Candidatus Photodesmus katoptron thioredoxin (CPk) and the CPk chimeras (CPk and CPk [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52]) were synthesized with a His-tag at the Cterminal and codon optimized for expression in E. coli cells. Mutations required for the single-mutant CPk variants (L7V, D10E, S11N, L14Q, N15E, I17L, S18K, A19S, S20D, G21K, V22P, F70Y, G74S, V75I, S77T), the double-mutant S11N/G74S variant and the chimera involving the loop [70][71][72][73][74][75][76][77] were introduced using the QuikChange Lighting Site-Directed Mutagenesis kit (Agilent Technologies) and the sequences were confirmed by DNA sequencing.…”

“…Finally, a somewhat intriguing result of our extensive experimental study on the stability of CPk thioredoxin variants deserves some attention. While most of the variants show the expected stability enhancement, this is not the case with CPk- [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] thioredoxin. This modern/ancestral chimera involves 10 back-to-ancestor amino acid replacements in the longest a-helix of the thioredoxin structure, which is expected to fold early according to our statistical-mechanical calculations (Fig.…”

“…4). CPk- [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] thioredoxin displays a stability similar to that of the CPk thioredoxin background as shown by both the denaturation temperature values and the chemical denaturation profiles (Fig. S3).…”

“…Examples include: phosphate-binding protein 22 , periplasmic binding protein 23 , serum paraoxonase 24 , coagulation factor VIII 25 , titin 26 , haloalkane dehalogenases 27 , cytidine and adenine base editors 28 , diterpene cyclase 29 , rubisco 30 , endoglucanases 31 , L-amino acid oxidases 32 , laccases 33 , front-end D6-desaturases 34 and fatty acid photo-decarboxylases 35 . On a related note, recent studies on ancestral proteins have noted an improved capability to yield crystals suitable for X-ray structural determination 36,37 .…”