Discovery, biochemical characterization, and bioengineering of cyanobactin prenyltransferases.

Prenylation is a post-translational modification (PTM) widely found in primary and secondary metabolism. This modification can enhance the lipophilicity of molecules, enabling them to interact with lipid membranes more effectively. The prenylation of peptides is often carried out by cyanobactin prenyltransferases (PTases) from cyanobacteria. These enzymes are of interest due to their ability to add prenyl groups to unmodified peptides, thus making them more effective therapeutics through the subsequent acquisition of increased membrane permeability and bioavailability. Herein we review the current knowledge of cyanobactin PTases, focusing on their discovery, biochemistry, and bioengineering, and highlight the potential application of them as peptide alkylation biocatalysts to generate peptide therapeutics.

Characterization of the N5-dimethylallyl-FMN Intermediate in the Biosynthesis of Prenylated-FMN Catalyzed by UbiX.

Prenylated-FMN (prFMN) is the cofactor used by the UbiD-like family of decarboxylases that catalyzes the decarboxylation of various aromatic and unsaturated carboxylic acids. prFMN is synthesized from reduced FMN and dimethylallyl phosphate (DMAP) by a specialized prenyl transferase, UbiX. UbiX catalyzes the sequential formation of two bonds, the first between N5 of the flavin and C1 of DMAP, and the second between C6 of the flavin and C3 of DMAP. We have examined the reaction of UbiX with both FMN and riboflavin. Although UbiX converts FMN to prFMN, we show that significant amounts of the N5-dimethylallyl-FMN intermediate are released from the enzyme during catalysis. With riboflavin as the substrate, UbiX catalyzes only a partial reaction, resulting in only N5-dimethylallyl-riboflavin being formed. Purification of the N5-dimethylallyl-FMN adduct allowed its structure to be verified by 1H NMR spectroscopy and its reactivity to be investigated. Surprisingly, whereas reduced prFMN oxidizes in seconds to form the stable prFMN semiquinone radical when exposed to air, N5-dimethylallyl-FMN oxidizes much more slowly over several hours; in this case, oxidation is accompanied by spontaneous hydrolysis to regenerate FMN. These studies highlight the important contribution that cyclization of the prenyl-derived ring of prFMN makes to the cofactor's biological activity.

Discovery, Characterization and Engineering of the Free l-Histidine C4-Prenyltransferase.

Prenylation of amino acids is a critical step for synthesizing building blocks of prenylated alkaloid family natural products, where the corresponding prenyltransferase that catalyzes prenylation on free l-histidine (l-His) has not yet been identified. Here, we first discovered and characterized a prenyltransferase FunA from the antifungal agent fungerin pathway that efficiently performs C4-dimethylallylation on l-His. Crystal structure-guided engineering of the prenyl-binding pocket of FunA, a single M181A mutation, successfully converted it into a C4-geranyltransferase. Furthermore, FunA and its variant FunA-M181A show broad substrate promiscuity toward substrates that vary in substituents of the imidazole ring. Our work furthers our knowledge of free amino acid prenyltransferase and expands the arsenal of alkylation biocatalysts for imidazole-containing small molecules.

Structural insights into the diverse prenylating capabilities of DMATS prenyltransferases.

Covering: 2009 up to August 2023Prenyltransferases (PTs) are involved in the primary and the secondary metabolism of plants, bacteria, and fungi, and they are key enzymes in the biosynthesis of many clinically relevant natural products (NPs). The continued biochemical and structural characterization of the soluble dimethylallyl tryptophan synthase (DMATS) PTs over the past two decades have revealed the significant promise that these enzymes hold as biocatalysts for the chemoenzymatic synthesis of novel drug leads. This is a comprehensive review of DMATSs describing the structure-function relationships that have shaped the mechanistic underpinnings of these enzymes, as well as the application of this knowledge to the engineering of DMATSs. We summarize the key findings and lessons learned from these studies over the past 14 years (2009-2023). In addition, we identify current gaps in our understanding of these fascinating enzymes.

A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus.

Protein prenylation is essential for many cellular processes including signal transduction, cytoskeletal reorganization, and membrane trafficking. Here, we identify a novel type of protein prenyltransferase, which we named geranylgeranyltransferase type-III (GGTase-III). GGTase-III consists of prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the ß subunit of RabGGTase. Using a biotinylated geranylgeranyl analogue, we identified the Golgi SNARE protein Ykt6 as a substrate of GGTase-III. GGTase-III transfers a geranylgeranyl group to mono-farnesylated Ykt6, generating doubly prenylated Ykt6. The crystal structure of GGTase-III in complex with Ykt6 provides structural basis for Ykt6 double prenylation. In GGTase-III-deficient cells, Ykt6 remained in a singly prenylated form, and the Golgi SNARE complex assembly was severely impaired. Consequently, the Golgi apparatus was structurally disorganized, and intra-Golgi protein trafficking was delayed. Our findings reveal a fourth type of protein prenyltransferase that generates geranylgeranyl-farnesyl Ykt6. Double prenylation of Ykt6 is essential for the structural and functional organization of the Golgi apparatus.

Substrate-Dependent Alteration in the C- and O-Prenylation Specificities of Cannabis Prenyltransferase.

CsPT4 is an aromatic prenyltransferase that synthesizes cannabigerolic acid (CBGA), the key intermediate of cannabinoid biosynthesis in Cannabis sativa, from olivetolic acid (OA) and geranyl diphosphate (GPP). CsPT4 has a catalytic potential to produce a variety of CBGA analogs via regioselective C-prenylation of aromatic substrates having resorcylic acid skeletons including bibenzyl 2,4-dihydroxy-6-phenylethylbenzoic acid (DPA). In this study, we further investigated the substrate specificity of CsPT4 using phlorocaprophenone (PCP) and 2',4',6'-trihydroxydihydrochalcone (THDC), the isomers of OA and DPA, respectively, and demonstrated that CsPT4 catalyzed both C-prenylation and O-prenylation reactions on PCP and THDC that share acylphloroglucinol substructures. Interestingly, the kinetic parameters of CsPT4 for these substrates differed depending on whether they underwent C-prenylation or O-prenylation, suggesting that this enzyme utilized different substrate-binding modes suitable for the respective reactions. Aromatic prenyltransferases that catalyze O-prenylation are rare in the plant kingdom, and CsPT4 was notable for altering the reaction specificity between C- and O-prenylations depending on the skeletons of aromatic substrates. We also demonstrated that enzymatically synthesized geranylated acylphloroglucinols had potent antiausterity activity against PANC-1 human pancreatic cancer cells, with 4'-O-geranyl THDC being the most effective. We suggest that CsPT4 is a valuable catalyst to generate biologically active C- and O-prenylated molecules that could be anticancer lead compounds.

De Novo Discovery of Pseudo-Natural Prenylated Macrocyclic Peptide Ligands.

Prenylation of peptides is widely observed in the secondary metabolites of diverse organisms, granting peptides unique chemical properties distinct from proteinogenic amino acids. Discovery of prenylated peptide agents has largely relied on isolation or genome mining of naturally occurring molecules. To devise a platform technology for de novo discovery of artificial prenylated peptides targeting a protein of choice, here we have integrated the thioether-macrocyclic peptide (teMP) library construction/selection technology, so-called RaPID (Random nonstandard Peptides Integrated Discovery) system, with a Trp-C3-prenyltransferase KgpF involved in the biosynthesis of a prenylated natural product. This unique enzyme exhibited remarkably broad substrate tolerance, capable of modifying various Trp-containing teMPs to install a prenylated residue with tricyclic constrained structure. We constructed a vast library of prenylated teMPs and subjected it to in vitro selection against a phosphoglycerate mutase. This selection platform has led to the identification of a pseudo-natural prenylated teMP inhibiting the target enzyme with an IC50 of 30 nM. Importantly, the prenylation was essential for the inhibitory activity, enhanced serum stability, and cellular uptake of the peptide, highlighting the benefits of peptide prenylation. This work showcases the de novo discovery platform for pseudo-natural prenylated peptides, which is readily applicable to other drug targets.

Switching Prenyl Donor Specificities of Cyanobactin Prenyltransferases.

Prenyltransferases in cyanobactin biosynthesis are of growing interest as peptide alkylation biocatalysts, but their prenylation modes characterized so far have been limited to dimethylallylation (C5) or geranylation (C10). Here we engaged in structure-guided engineering of the prenyl-binding pocket of a His-C2-geranyltransferase LimF to modulate its prenylation mode. Contraction of the pocket by a single mutation led to a His-C2-dimethylallyltransferase. More importantly, pocket expansion by a double mutation successfully repurposed LimF for farnesylation (C15), which is an unprecedented mode in this family. Furthermore, the obtained knowledge of the essential residues to construct the farnesyl-binding pocket has allowed for rational design of a Tyr-O-farnesyltransferase by a triple mutation of a Tyr-O-dimethylallyltransferase PagF. These results provide an approach to manipulate the prenyl specificity of cyanobactin prenyltransferases, broadening the chemical space covered by this class of enzymes and expanding the toolbox of peptide alkylation biocatalysts.

Generation of Cannabigerolic Acid Derivatives and Their Precursors by Using the Promiscuity of the Aromatic Prenyltransferase NphB.

NphB is an aromatic prenyltransferase with high promiscuity for phenolics including flavonoids, isoflavonoids, and plant polyketides. It has been demonstrated that cannabigerolic acid is successfully formed by the reaction catalysed by NphB using geranyl diphosphate and olivetolic acid as substrates. In this study, the substrate specificity of NphB was further determined by using olivetolic acid derivatives as potential substrates for the formation of new synthetic cannabinoids. The derivatives differ in the hydrocarbon chain attached to C6 of the core structure. We performed in silico experiments, including docking of olivetolic acid derivatives, to identify differences in their binding modes. Substrate acceptance was predicted. Based on these results, a library of olivetolic acid derivatives was constructed and synthesized by using different organic synthetic routes. Conversion was monitored in in vitro assays with purified NphB versions. For the substrates leading to a high conversion olivetolic acid-C8, olivetolic acid-C2 and 2-benzyl-4,6-dihydroxybenzoic acid, the products were further elucidated and identified as cannbigerolic acid derivatives. Therefore, these substrates show potential to be adapted in cannabinoid biosynthesis.

Catalysts for the Enzymatic Lipidation of Peptides.

Biologically active peptides are a major growing class of drugs, but their therapeutic potential is constrained by several limitations including bioavailability and poor pharmacokinetics. The attachment of functional groups like lipids has proven to be a robust and effective strategy for improving their therapeutic potential. Biochemical and bioactivity-guided screening efforts have identified the cyanobactins as a large class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that are modified with lipids. These lipids are attached by the F superfamily of peptide prenyltransferase enzymes that utilize 5-carbon (prenylation) or 10-carbon (geranylation) donors. The chemical structures of various cyanobactins initially showed isoprenoid attachments on Ser, Thr, or Tyr. Biochemical characterization of the F prenyltransferases from the corresponding clusters shows that the different enzymes have different acceptor residue specificities but are otherwise remarkably sequence tolerant. Hence, these enzymes are well suited for biotechnological applications. The crystal structure of the Tyr O-prenyltransferase PagF reveals that the F enzyme shares a domain architecture reminiscent of a canonical ABBA prenyltransferase fold but lacks secondary structural elements necessary to form an enclosed active site. Binding of either cyclic or linear peptides is sufficient to close the active site to allow for productive catalysis, explaining why these enzymes cannot use isolated amino acids as substrates.Almost all characterized isoprenylated cyanobactins are modified with 5-carbon isoprenoids. However, chemical characterization demonstrates that the piricyclamides are modified with a 10-carbon geranyl moiety, and in vitro reconstitution of the corresponding PirF shows that the enzyme is a geranyltransferase. Structural analysis of PirF shows an active site nearly identical with that of the PagF prenyltransferase but with a single amino acid substitution. Of note, mutation at this residue in PagF or PirF can completely switch the isoprenoid donor specificity of these enzymes. Recent efforts have resulted in significant expansion of the F family with enzymes identified that can carry out C-prenylations of Trp, N-prenylations of Trp, and bis-N-prenylations of Arg. Additional genome-guided efforts based on the sequence of F enzymes identify linear cyanobactins that are α-N-prenylated and α-C-methylated by a bifunctional prenyltransferase/methyltransferase fusion and a bis-α-N- and α-C-prenylated linear peptide. The discovery of these different classes of prenyltransferases with diverse acceptor residue specificities expands the biosynthetic toolkit for enzymatic prenylation of peptide substrates.In this Account, we review the current knowledge scope of the F family of peptide prenyltransferases, focusing on the biochemical, structure-function, and chemical characterization studies that have been carried out in our laboratories. These enzymes are easily amenable for diversity-oriented synthetic efforts as they can accommodate substrate peptides of diverse sequences and are thus attractive catalysts for use in synthetic biology approaches to generate high-value peptidic therapeutics.

Algal neurotoxin biosynthesis repurposes the terpene cyclase structural fold into an N-prenyltransferase.

Prenylation is a common biological reaction in all domains of life wherein prenyl diphosphate donors transfer prenyl groups onto small molecules as well as large proteins. The enzymes that catalyze these reactions are structurally distinct from ubiquitous terpene cyclases that, instead, assemble terpenes via intramolecular rearrangements of a single substrate. Herein, we report the structure and molecular details of a new family of prenyltransferases from marine algae that repurposes the terpene cyclase structural fold for the N-prenylation of glutamic acid during the biosynthesis of the potent neurochemicals domoic acid and kainic acid. We solved the X-ray crystal structure of the prenyltransferase found in domoic acid biosynthesis, DabA, and show distinct active site binding modifications that remodel the canonical magnesium (Mg2+)-binding motif found in terpene cyclases. We then applied our structural knowledge of DabA and a homologous enzyme from the kainic acid biosynthetic pathway, KabA, to reengineer their isoprene donor specificities (geranyl diphosphate [GPP] versus dimethylallyl diphosphate [DMAPP]) with a single amino acid change. While diatom DabA and seaweed KabA enzymes share a common evolutionary lineage, they are distinct from all other terpene cyclases, suggesting a very distant ancestor to the larger terpene synthase family.

Structural Basis of Substrate Promiscuity and Catalysis by the Reverse Prenyltransferase N-Dimethylallyl-l-tryptophan Synthase from Fusarium fujikuroi.

The regiospecific prenylation of an aromatic amino acid catalyzed by a dimethylallyl-l-tryptophan synthase (DMATS) is a key step in the biosynthesis of many fungal and bacterial natural products. DMATS enzymes share a common "ABBA" fold with divergent active site contours that direct alternative C-C, C-N, and C-O bond-forming trajectories. DMATS1 from Fusarium fujikuroi catalyzes the reverse N-prenylation of l-Trp by generating an allylic carbocation from dimethylallyl diphosphate (DMAPP) that then alkylates the indole nitrogen of l-Trp. DMATS1 stands out among the greater DMATS family because it exhibits unusually broad substrate specificity: it can utilize geranyl diphosphate (GPP) or l-Tyr as an alternative prenyl donor or acceptor, respectively; it can catalyze both forward and reverse prenylation, i.e., at C1 or C3 of DMAPP; and it can catalyze C-N and C-O bond-forming reactions. Here, we report the crystal structures of DMATS1 and its complexes with l-Trp or l-Tyr and unreactive thiolodiphosphate analogues of the prenyl donors DMAPP and GPP. Structures of ternary complexes mimic Michaelis complexes with actual substrates and illuminate active site features that govern prenylation regiochemistry. Comparison with CymD, a bacterial enzyme that catalyzes the reverse N-prenylation of l-Trp with DMAPP, indicates that bacterial and fungal DMATS enzymes share a conserved reaction mechanism. However, the narrower active site contour of CymD enforces narrower substrate specificity. Structure-function relationships established for DMATS enzymes will ultimately inform protein engineering experiments that will broaden the utility of these enzymes as useful tools for synthetic biology.

Data Science-Driven Analysis of Substrate-Permissive Diketopiperazine Reverse Prenyltransferase NotF: Applications in Protein Engineering and Cascade Biocatalytic Synthesis of (-)-Eurotiumin A.

Prenyltransfer is an early-stage carbon-hydrogen bond (C-H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Toward the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). As the first tailoring event within the biosynthesis of cytotoxic notoamide metabolites, PT NotF catalyzes C2 reverse prenyltransfer of brevianamide F. Solving a crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent-exposed active site, intimating NotF may possess a significantly broad substrate scope. To assess the substrate selectivity of NotF, we synthesized a panel of 30 sterically and electronically differentiated tryptophanyl DKPs, the majority of which were selectively prenylated by NotF in synthetically useful conversions (2 to >99%). Quantitative representation of this substrate library and development of a descriptive statistical model provided insight into the molecular origins of NotF's substrate promiscuity. This approach enabled the identification of key substrate descriptors (electrophilicity, size, and flexibility) that govern the rate of NotF-catalyzed prenyltransfer, and the development of an "induced fit docking (IFD)-guided" engineering strategy for improved turnover of our largest substrates. We further demonstrated the utility of NotF in tandem with oxidative cyclization using flavin monooxygenase, BvnB. This one-pot, in vitro biocatalytic cascade enabled the first chemoenzymatic synthesis of the marine fungal natural product, (-)-eurotiumin A, in three steps and 60% overall yield.

Structure, catalysis, and inhibition mechanism of prenyltransferase.

Isoprenoids, also known as terpenes or terpenoids, represent a large family of natural products composed of five-carbon isopentenyl diphosphate or its isomer dimethylallyl diphosphate as the building blocks. Isoprenoids are structurally and functionally diverse and include dolichols, steroid hormones, carotenoids, retinoids, aromatic metabolites, the isoprenoid side-chain of ubiquinone, and isoprenoid attached signaling proteins. Productions of isoprenoids are catalyzed by a group of enzymes known as prenyltransferases, such as farnesyltransferases, geranylgeranyltransferases, terpenoid cyclase, squalene synthase, aromatic prenyltransferase, and cis- and trans-prenyltransferases. Because these enzymes are key in cellular processes and metabolic pathways, they are expected to be potential targets in new drug discovery. In this review, six distinct subsets of characterized prenyltransferases are structurally and mechanistically classified, including (1) head-to-tail prenyl synthase, (2) head-to-head prenyl synthase, (3) head-to-middle prenyl synthase, (4) terpenoid cyclase, (5) aromatic prenyltransferase, and (6) protein prenylation. Inhibitors of those enzymes for potential therapies against several diseases are discussed. Lastly, recent results on the structures of integral membrane enzyme, undecaprenyl pyrophosphate phosphatase, are also discussed.

UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis.

Ubiquinone (also known as coenzyme Q) is a ubiquitous lipid-soluble redox cofactor that is an essential component of electron transfer chains. Eleven genes have been implicated in bacterial ubiquinone biosynthesis, including ubiX and ubiD, which are responsible for decarboxylation of the 3-octaprenyl-4-hydroxybenzoate precursor. Despite structural and biochemical characterization of UbiX as a flavin mononucleotide (FMN)-binding protein, no decarboxylase activity has been detected. Here we report that UbiX produces a novel flavin-derived cofactor required for the decarboxylase activity of UbiD. UbiX acts as a flavin prenyltransferase, linking a dimethylallyl moiety to the flavin N5 and C6 atoms. This adds a fourth non-aromatic ring to the flavin isoalloxazine group. In contrast to other prenyltransferases, UbiX is metal-independent and requires dimethylallyl-monophosphate as substrate. Kinetic crystallography reveals that the prenyltransferase mechanism of UbiX resembles that of the terpene synthases. The active site environment is dominated by π systems, which assist phosphate-C1' bond breakage following FMN reduction, leading to formation of the N5-C1' bond. UbiX then acts as a chaperone for adduct reorientation, via transient carbocation species, leading ultimately to formation of the dimethylallyl C3'-C6 bond. Our findings establish the mechanism for formation of a new flavin-derived cofactor, extending both flavin and terpenoid biochemical repertoires.

Genome-Mining-Based Discovery of the Cyclic Peptide Tolypamide and TolF, a Ser/Thr Forward O-Prenyltransferase.

Cyanobactins comprise a widespread group of peptide metabolites produced by cyanobacteria that are often diversified by post-translational prenylation. Several enzymes have been identified in cyanobactin biosynthetic pathways that carry out chemically diverse prenylation reactions, representing a resource for the discovery of post-translational alkylating agents. Here, genome mining was used to identify orphan cyanobactin prenyltransferases, leading to the isolation of tolypamide from the freshwater cyanobacterium Tolypothrix sp. The structure of tolypamide was confirmed by spectroscopic methods, degradation, and enzymatic total synthesis. Tolypamide is forward-prenylated on a threonine residue, representing an unprecedented post-translational modification. Biochemical characterization of the cognate enzyme TolF revealed a prenyltransferase with strict selectivity for forward O-prenylation of serine or threonine but with relaxed substrate selectivity for flanking peptide sequences. Since cyanobactin pathways often exhibit exceptionally broad substrate tolerance, these enzymes represent robust tools for synthetic biology.

Bringing Bioactive Compounds into Membranes: The UbiA Superfamily of Intramembrane Aromatic Prenyltransferases.

The UbiA superfamily of intramembrane prenyltransferases catalyzes a key biosynthetic step in the production of ubiquinones, menaquinones, plastoquinones, hemes, chlorophylls, vitamin E, and structural lipids. These lipophilic compounds serve as electron and proton carriers for cellular respiration and photosynthesis, as antioxidants to reduce cell damage, and as structural components of microbial cell walls and membranes. This article reviews the biological functions and enzymatic activities of representative members of the superfamily, focusing on the remarkable recent research progress revealing that the UbiA superfamily is centrally implicated in several important physiological processes and human diseases. Because prenyltransferases in this superfamily have distinctive substrate preferences, two recent crystal structures are compared to illuminate the general mechanism for substrate recognition.

Terpene synthases in disguise: enzymology, structure, and opportunities of non-canonical terpene synthases.

Covering: up to July 2019 Terpene synthases (TSs) are responsible for generating much of the structural diversity found in the superfamily of terpenoid natural products. These elegant enzymes mediate complex carbocation-based cyclization and rearrangement cascades with a variety of electron-rich linear and cyclic substrates. For decades, two main classes of TSs, divided by how they generate the reaction-triggering initial carbocation, have dominated the field of terpene enzymology. Recently, several novel and unconventional TSs that perform TS-like reactions but do not resemble canonical TSs in sequence or structure have been discovered. In this review, we identify 12 families of non-canonical TSs and examine their sequences, structures, functions, and proposed mechanisms. Nature provides a wide diversity of enzymes, including prenyltransferases, methyltransferases, P450s, and NAD+-dependent dehydrogenases, as well as completely new enzymes, that utilize distinctive reaction mechanisms for TS chemistry. These unique non-canonical TSs provide immense opportunities to understand how nature evolved different tools for terpene biosynthesis by structural and mechanistic characterization while affording new probes for the discovery of novel terpenoid natural products and gene clusters via genome mining. With every new discovery, the dualistic paradigm of TSs is contradicted and the field of terpene chemistry and enzymology continues to expand.

Reinvestigation of the substrate specificity of a reverse prenyltransferase NotF from Aspergillus sp. MF297-2.

NotF from Aspergillus sp. MF297-2 and BrePT from Aspergillus versicolor catalyze a reverse C2-prenylation of brevianamide F in the biosynthetic pathway of brevianamides and notoamides. NotF was reported to use only brevianamide F as substrate while BrePT demonstrated broad substrate promiscuity. With high identity at amino acid level, it is interesting to reinvestigate the catalytic activities of these two prenyltransferases in vitro toward 14 cyclodipeptides. Product identification of the in vitro assays by MS proved that NotF and BrePT share similar catalytic ability and substrate promiscuity.

Acceptor substrate determines donor specificity of an aromatic prenyltransferase: expanding the biocatalytic potential of NphB.

Aromatic prenyltransferases are known for their extensive promiscuity toward aromatic acceptor substrates and their ability to form various carbon-carbon and carbon-heteroatom bonds. Of particular interest among the prenyltransferases is NphB, whose ability to geranylate cannabinoid precursors has been utilized in several in vivo and in vitro systems. It has therefore been established that prenyltransferases can be utilized as biocatalysts for the generation of useful compounds. However, recent observations of non-native alkyl-donor promiscuity among prenyltransferases indicate the role of NphB in biocatalysis could be expanded beyond geranylation reactions. Therefore, the goal of this study was to elucidate the donor promiscuity of NphB using different acceptor substrates. Herein, we report distinct donor profiles between NphB-catalyzed reactions involving the known substrate 1,6-dihydroxynaphthalene and an FDA-approved drug molecule sulfabenzamide. Furthermore, we report the first instance of regiospecific, NphB-catalyzed N-alkylation of sulfabenzamide using a library of non-native alkyl-donors, indicating the biocatalytic potential of NphB as a late-stage diversification tool. KEY POINTS: • NphB can utilize the antibacterial drug sulfabenzamide as an acceptor. • The donor profile of NphB changes dramatically with the choice of acceptor. • NphB performs a previously unknown regiospecific N-alkylation on sulfabenzamide. • Prenyltransferases like NphB can be utilized as drug-alkylating biocatalysts.