Experimental studies support that protein engineering based on ancestral sequence reconstruction often leads to variants with biotechnologically useful biomolecular properties. These may include high stability, enhanced conformational flexibility and a modified catalysis range. Carbohydrate-active enzymes have numerous applications related to the degradation and synthesis of carbohydrates and glycoconjugates. Herein, we explore how ancestral reconstruction impacts the substrate scope of glycosidases, highly diverse enzymes that catalyze the hydrolysis of glycosidic bonds in all living cells and find applications as catalysts of the synthetic reaction. To this end, we screened 291 potential glycosidase substrates for degradation by both a modern family-1 glycosidase from the halothermophilic bacterium Halothermothrix orenii and a putative ancestral family-1 glycosidase derived from sequence reconstruction of a bacterial-eukaryotic common ancestor. The modern enzyme is the better catalyst for many substrates, but the ancestral glycosidase is more efficient with flavonoid glycosides bearing large-aglycone moieties. Analysis of the catalytic parameters for a selected set of substrates, alongside analysis of the library data using a supervised learning algorithm, supports the hypothesis that the modern enzyme tends to become less catalytically efficient with increasing substrate size, while this trend is not observed for the ancestral glycosidase. Molecular modeling supports that the ancestral catalysis pattern is linked to the existence of a highly flexible region of the protein and a cavity capable of accommodating large aglycones. Our results and analyses provide guidelines for the engineering of enzymes for the synthesis and hydrolysis of large glycoconjugates.

Many natural products and pharmaceutical compounds bear the pyrroloindoline scaffold, highlighting the importance of the heterocyclic motif. Here, we aim at expanding the toolset for the selective synthesis of pyrroloindolines by characterising and employing the N-methyltransferase SgPsmC from Streptomyces griseofuscus, an enzyme involved in the biosynthesis of physostigmine, in a selective kinetic resolution of pyrroloindolines performed at a laboratory preparative scale.

Promiscuity, the capability of catalyzing a diversity of chemical reactions, is a desirable feature in many enzymes intended for biotechnological applications. Promiscuous activities, however, are typically depressed in specialized modern enzymes. On the other hand, several ancestral reconstruction studies have reported enzymes with enhanced promiscuity. Glycosidases catalyze the hydrolysis of glycosidic bonds in all living cells but find practical applications in the synthesis of glycoconjugates as catalysts of the reverse reaction. Here, we use a library of ∼ 500 possible substrates to compare the catalytic scope of the modern family-1 glycosidase from Halothermothrix orenii with that of a putative ancestral family-1 glycosidase derived from sequence reconstruction at a bacterial-eukaryotic common ancestor. The modern enzyme is the better catalyst for the hydrolysis of small substrates typically used to assess the activity of family-1 glycosidases. We identify, however, a trend for the modern enzyme to become less catalytically efficient with increasing substrate size. Such trend is not apparent in the conformationally-flexible ancestral glycosidase, which is in fact the better catalyst for the hydrolysis of flavonoid glycosides. Our results support that ancestral sequence reconstruction may provide a basis for enzyme engineering for the synthesis of glycoconjugates with large glycosyl acceptors. ### Competing Interest Statement Leonardo De Maria, Martin Hayes and Francesco Falcioni are current employees and/or shareholders of AstraZeneca. The authors declare no competing interest.

Enzyme-catalyzed late-stage functionalization (LSF), such as methylation of drug molecules and lead structures, enables direct access to more potent active pharmaceutical ingredients (API). S-adenosyl-l-methionine-dependent methyltransferases (MTs) can play a key role in the development of new APIs, as they catalyze the chemo- and regioselective methylation of O-, N-, S- and C-atoms, being superior to traditional chemical routes. To identify suitable MTs, we developed a continuous fluorescence-based, high-throughput assay for SAM-dependent methyltransferases, which facilitates screening using E. coli cell lysates. This assay involves two enzymatic steps for the conversion of S-adenosyl-l-homocysteine into H2 S to result in a selective fluorescence readout via reduction of an azidocoumarin sulfide probe. Investigation of two O-MTs and an N-MT confirmed that this assay is suitable for the determination of methyltransferase activity in E. coli cell lysates.

Even though pyrroloindoles are widely present in natural products with different kinds of biological activities, their selective synthesis remains challenging with existing tools in organic chemistry, and there is furthermore a demand for stereoselective and mild methods to access this structural motif. Nature uses C3-methyltransferases to form the pyrroloindole framework, starting from the amino acid tryptophan. In the present study, the SAM-dependent methyltransferase StspM1 from Streptomyces sp. HPH0547 is used to build the pyrroloindole structural motif in tryptophan-based diketopiperazines (DKP). The substrate scope of the enzyme regarding different Trp-Trp-DKP isomers was investigated on an experimental and computational level. After further characterization and optimization of the methylation reaction with a design of experiment approach, a preparative scale reaction with the immobilized enzyme including a SAM regeneration system was performed to show the synthetic use of this biocatalytic tool to access the pyrroloindole structural motif.

AbstractDie enzymkatalysierte Funktionalisierung (engl.: late‐stage functionalization (LSF)), wie z. B. die Methylierung von Arzneimittelmolekülen und Leitstrukturen, ermöglicht den direkten Zugang zu wirksameren pharmazeutischen Wirkstoffen (API). S‐Adenosyl‐l‐Methionin‐abhängige Methyltransferasen (MTs) können eine Schlüsselrolle bei der Entwicklung neuer Wirkstoffe spielen, da sie die chemo‐ und regioselektive Methylierung von O‐, N‐, S‐ und C‐Atomen katalysieren und damit den herkömmlichen chemischen Wegen überlegen sind. Um geeignete MTs zu identifizieren, haben wir einen kontinuierlichen fluoreszenzbasierten Hochdurchsatzassay für SAM‐abhängige Methyltransferasen entwickelt, der das Screening unter Verwendung von E. coli Zelllysaten erleichtert. Dieser Assay besteht aus zwei enzymatischen Schritten für die Umwandlung von S‐Adenosyl‐l‐Homocystein zu H2S, was zu einer selektiven Fluoreszenzmessung durch Reduktion eines Azidocoumarin‐Sulfidsensors führt. Die Untersuchung von zwei O‐MTs und einer N‐MT bestätigte, dass dieser Assay für die Bestimmung der Methyltransferase‐Aktivität in E. coli Zelllysaten geeignet ist.

Enantioselective methylation is a challenging task in organic chemistry, yet often desirable in drug discovery and optimization. S-Adenosyl methionine (SAM)-dependent methyltransferases (MTases) offer a selective alternative to chemical synthesis and an abundance of potential scaffolds. The crystal structure of C3-indole MTase PsmD from Streptomyces griseofuscus, involved in the biosynthesis of the acetylcholinesterase inhibitor physostigmine, was determined via X-ray crystallography. The amino acid residues essential for catalysis were identified by site-directed mutagenesis, and a mechanism of action was proposed. Furthermore, a PsmD ortholog was identified and characterized. The variant catalyzed enantioselective C-methylation over a broad substrate scope while displaying increased stability. Using this enzyme, preparative-scale enzymatic methylation was performed in cell-free extracts in combination with an SAM recycling system, eliminating the need for cofactor supplementation.

AbstractDie enantioselektive Synthese bioaktiver Verbindungen, die ein Pyrroloindolgerüst tragen, ist oft aufwändig. Im Gegensatz dazu sind mehrere S‐Adenosylmethionin (SAM)‐abhängige Methyltransferasen bekannt, die stereo‐ und regioselektive Methylierungen an der C3‐Position verschiedener Indole durchführen und damit eine direkte Bildung des gewünschten Pyrroloindolgerüsts ermöglichen. Hier wird die SAM‐abhängige Methyltransferase PsmD aus Streptomyces griseofuscus, ein Schlüsselenzym in der Biosynthese von Physostigmin, im Detail charakterisiert. Die biochemischen Eigenschaften von PsmD und ihr Substratspektrum werden aufgezeigt. Die enzymatische Methylierung im präparativen Maßstab, einschließlich der SAM‐Regeneration, wurde für drei ausgewählte Substrate nach einer Design‐of‐Experiment‐Optimierung erreicht.

For more than two decades, the development of potent acetylcholinesterase (AChE) inhibitors has been an ongoing task to treat dementia associated with Alzheimer’s disease and improve the pharmacokinetic properties of existing drugs. In the present study, we used three docking-based virtual screening approaches to screen both ZINC15 and MolPort databases for synthetic analogs of physostigmine and donepezil, two highly potent AChE inhibitors. We characterised the in vitro inhibitory concentration of 11 compounds, ranging from 14 to 985 μM. The most potent of these compounds, S-I 26, showed a fivefold improved inhibitory concentration in comparison to rivastigmine. Moderate inhibitors carrying novel scaffolds were identified and could be improved for the development of new classes of AChE inhibitors.

The alkaloid physostigmine is an approved anticholinergic drug and an important lead structure for the development of novel therapeutics. Using a complementary approach that merged chemical synthesis with pathway refactoring, we produced a series of physostigmine analogues with altered specificity and toxicity profiles in the heterologous host Myxococcus xanthus. The compounds that were generated by applying a simple feeding strategy include the promising drug candidate phenserine, which was previously accessible only by total synthesis.

Enantioselective synthesis of bioactive compounds bearing a pyrroloindole framework is often laborious. In contrast, there are several S-adenosyl methionine (SAM)-dependent methyl transferases known for stereo- and regioselective methylation at the C3 position of various indoles, directly leading to the formation of the desired pyrroloindole moiety. Herein, the SAM-dependent methyl transferase PsmD from Streptomyces griseofuscus, a key enzyme in the biosynthesis of physostigmine, is characterized in detail. The biochemical properties of PsmD and its substrate scope were demonstrated. Preparative scale enzymatic methylation including SAM regeneration was achieved for three selected substrates after a design-of-experiment optimization.