Molecular Basis for the Substrate Promiscuity of Isopentenyl Phosphate Kinase from Candidatus methanomethylophilus alvus.

Isopentenyl phosphate kinases (IPKs) catalyze the ATP-dependent phosphorylation of isopentenyl monophosphate (IP) to isopentenyl diphosphate (IPP) in the alternate mevalonate pathways of the archaea and plant cytoplasm. In recent years, IPKs have also been employed in artificial biosynthetic pathways called "(iso) prenol pathways" that utilize promiscuous kinases to sequentially phosphorylate (iso) prenol and generate the isoprenoid precursors IPP and dimethylallyl diphosphate (DMAPP). Furthermore, IPKs have garnered attention for their impressive substrate promiscuity toward non-natural alkyl-monophosphates (alkyl-Ps), which has prompted their utilization as biocatalysts for the generation of novel isoprenoids. However, none of the IPK crystal structures currently available contain non-natural substrates, leaving the roles of active-site residues in substrate promiscuity ambiguous. To address this, we present herein the high-resolution crystal structures of an IPK from Candidatus methanomethylophilus alvus (CMA) in the apo form and bound to natural and non-natural substrates. Additionally, we describe active-site engineering studies leading to enzyme variants with broadened substrate scope, as well as structure determination of two such variants (Ile74Ala and Ile146Ala) bound to non-natural alkyl-Ps. Collectively, our crystallographic studies compare six structures of CMA variants in different ligand-bound forms and highlight contrasting structural dynamics of the two substrate-binding sites. Furthermore, the structural and mutational studies confirm a novel role of the highly conserved DVTGG motif in catalysis, both in CMA and in IPKs at large. As such, the current study provides a molecular basis for the substrate-binding modes and catalytic performance of CMA toward the goal of developing IPKs into useful biocatalysts.

Chemoenzymatic synthesis of daptomycin analogs active against daptomycin-resistant strains.

Daptomycin is a last resort antibiotic for the treatment of infections caused by many Gram-positive bacterial strains, including vancomycin-resistant Enterococcus (VRE) and methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA). However, the emergence of daptomycin-resistant strains of S. aureus and Enterococcus in recent years has renewed interest in synthesizing daptomycin analogs to overcome resistance mechanisms. Within this context, three aromatic prenyltransferases have been shown to accept daptomycin as a substrate, and the resulting prenylated analog was shown to be more potent against Gram-positive strains than the parent compound. Consequently, utilizing prenyltransferases to derivatize daptomycin offered an attractive alternative to traditional synthetic approaches, especially given the molecule's structural complexity. Herein, we report exploiting the ability of prenyltransferase CdpNPT to synthesize alkyl-diversified daptomycin analogs in combination with a library of synthetic non-native alkyl-pyrophosphates. The results revealed that CdpNPT can transfer a variety of alkyl groups onto daptomycin's tryptophan residue using the corresponding alkyl-pyrophosphates, while subsequent scaled-up reactions suggested that the enzyme can alkylate the N1, C2, C5, and C6 positions of the indole ring. In vitro antibacterial activity assays using 16 daptomycin analogs revealed that some of the analogs displayed 2-80-fold improvements in potency against MRSA, VRE, and daptomycin-resistant strains of S. aureus and Enterococcus faecalis. Thus, along with the new potent analogs, these findings have established that the regio-chemistry of alkyl substitution on the tryptophan residue can modulate daptomycin's potency. With additional protein engineering to improve the regio-selectivity, the described method has the potential to become a powerful tool for diversifying complex indole-containing molecules. KEY POINTS: • CdpNPT displays impressive donor promiscuity with daptomycin as the acceptor. • CdpNPT catalyzes N1-, C2-, C5-, and C6-alkylation on daptomycin's tryptophan residue. • Differential alkylation of daptomycin's tryptophan residue modulates its activity.

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.

FgaPT2, a biocatalytic tool for alkyl-diversification of indole natural products.

Aromatic prenyltransferases from natural product biosynthetic pathways display relaxed specificity for their aromatic substrates. While a growing body of evidence suggests aromatic prenyltransferases to be more tolerant towards their alkyl-donor substrates, most studies aimed at probing their donor-substrate specificity are limited to only a small set of alkyl pyrophosphate donors, restricting their broader utility as biocatalysts for synthetic applications. Here, we assess the donor substrate specificity of an l-tryptophan C4-prenyltransferase, also known as C4-dimethylallyltryptophan synthase, FgaPT2 from Aspergillus fumigatus, using an array of 34 synthetic unnatural alkyl-pyrophosphate analogues, and demonstrate FgaPT2 can catalyze the transfer of 25 of the 34 non-native alkyl groups from their corresponding synthetic alkyl-pyrophosphate analogues at N1, C3, C4 and C5 position of tryptophan in a normal and reverse manner. The kinetic studies and regio-chemical analysis of the alkyl-l-tryptophan products suggest that the alkyl-donor transfer by FgaPT2 is a function of the stability of the carbocation and the steric factors in the active site of the enzyme. Further, to demonstrate the biocatalytic utility of FgaPT2, this study also highlights the FgaPT2-catalyzed synthesis of a small set of alkyl-diversified indolocarbazole analogues. These results reveal FgaPT2 to be more tolerant to diverse non-native alkyl-donor substrates beyond their known acceptor substrate promiscuity and set the stage for its development as a novel biocatalytic tool for the differential alkylation of natural products for drug discovery and other synthetic applications.

Determination of Alkyl-Donor Promiscuity of Tyrosine-O-Prenyltransferase SirD from Leptosphaeria maculans.

Natural product prenyltransferases are known to display relaxed acceptor substrate specificity. Although recent studies with a small set of unnatural alkyl donors have revealed that prenyltransferases are flexible with regard to their alkyl donors, the scope of their alkyl donor specificity remains poorly understood. Towards this goal, we report the synthesis of 20 unnatural alkyl pyrophosphate donors and an assessment of the reactions of these synthetic unnatural alkyl pyrophosphate analogues catalyzed by tyrosine O-prenyltransferase SirD. This study demonstrates that SirD can utilize 16 out of 21 alkyl pyrophosphate analogues (including the natural donor) in catalyzing mostly O-alkylation of l-tyrosine. This study reveals the broad alkyl donor specificity of SirD and opens the door for the interrogation of the alkyl donor specificity of other prenyltransferases for potential utility as biocatalysts for differential alkylation applications.

Efficacy of ampicillin against methicillin-resistant Staphylococcus aureus restored through synergy with branched poly(ethylenimine).

ß-Lactam antibiotics kill Staphylococcus aureus bacteria by inhibiting the function of cell wall penicillin-binding proteins (PBPs) 1 and 3. However, ß-lactams are ineffective against PBP2a, used by methicillin-resistant S. aureus (MRSA) to perform essential cell wall crosslinking functions. PBP2a requires teichoic acid to properly locate and orient the enzyme, and thus MRSA is susceptible to antibiotics that prevent teichoic acid synthesis in the bacterial cytoplasm. As an alternative, we have used branched poly(ethylenimine), BPEI, to target teichoic acid in the bacterial cell wall. The result is restoration of MRSA susceptibility to the ß-lactam antibiotic ampicillin with a MIC of 1 µg ml-1, superior to that of vancomycin (MIC=3.7 µg ml-1). A checkerboard assay shows synergy of BPEI and ampicillin. NMR data show that BPEI alters the teichoic acid chemical environment. Laser scanning confocal microscopy images show BPEI residing on the bacterial cell wall, where teichoic acids and PBPs are located.