The utility of Streptococcus mutans undecaprenol kinase for the chemoenzymatic synthesis of diverse non-natural isoprenoids.

Isoprene chemoenzymatic cascades (ICCs) overcome the complexity of natural pathways by leveraging a streamlined two-enzyme cascade, facilitating efficient synthesis of C5-isoprene diphosphate precursors from readily available alcohol derivatives. Despite the documented promiscuity of enzymes in ICCs, exploration of their potential for accessing novel compounds remains limited, and existing methods require additional enzymes for generating longer-chain diphosphates. In this study, we present the utility of Streptococcus mutans undecaprenol kinase (SmUdpK) for the chemoenzymatic synthesis of diverse non-natural isoprenoids. Using a library of 50 synthetic alcohols, we demonstrate that SmUdpK's promiscuity extends to allylic chains as small as four carbons and benzylic alcohols with various substituents. Subsequently, SmUdpK is utilized in an ICC with isopentenyl phosphate kinase and aromatic prenyltransferase to generate multiple non-natural isoprenoids. This work provides evidence that, with proper optimization, SmUdpK can act as the first enzyme in these ICCs, enhancing access to both valuable and novel compounds.

Chemoenzymatic Modification of Daptomycin: Aromatic Group Installation on Trp1.

Daptomycin is a cyclic lipodepsipeptide antibiotic used to treat infections caused by Gram-positive pathogens, including multi-drug resistant strains such as methicillin-resistant Staphylococcus au-reus (MRSA) and vancomycin-resistant enterococci (VRE). The emergence of daptomycin-resistant bacterial strains has renewed interest in generating daptomycin analogs. Previous studies have shown that replacing the tryptophan of daptomycin with aromatic groups can generate analogs with enhanced potency. Additionally, we have demonstrated that aromatic prenyltransferases can attach diverse groups to the tryptophan of daptomycin. Here, we report the use of the prenyltransferase CdpNPT to derivatize the tryptophan of daptomycin with a library of benzylic and heterocyclic pyrophosphates. An analytical-scale study revealed that CdpNPT can transfer various aromatic groups onto daptomycin. Subsequent scaled-up and purified reactions indicated that the enzyme can attach aromatic groups to N1, C2, C5 and C6 positions of Trp1 of daptomycin. In vitro antibacterial activity assays using six of these purified compounds identified aromatic substituted daptomycin analogs show potency against both daptomycin-susceptible and resistant strains of Gram-positive bacteria. These findings suggest that installing aromatic groups on the Trp1 of daptomycin can lead to the generation of potent daptomycin analogs.

Ternary complexes of isopentenyl phosphate kinase from Thermococcus paralvinellae reveal molecular determinants of non-natural substrate specificity.

Isopentenyl phosphate kinases (IPKs) have recently garnered attention for their central role in biocatalytic "isoprenol pathways," which seek to reduce the synthesis of the isoprenoid precursors to two enzymatic steps. Furthermore, the natural promiscuity of IPKs toward non-natural alkyl-monophosphates (alkyl-Ps) as substrates has hinted at the isoprenol pathways' potential to access novel isoprenoids with potentially useful activities. However, only a handful of IPK crystal structures have been solved to date, and even fewer of these contain non-natural substrates bound in the active site. The current study sought to elucidate additional ternary complexes bound to non-natural substrates using the IPK homolog from Thermococcus paralvinellae (TcpIPK). Four such structures were solved, each bound to a different non-natural alkyl-P and the phosphoryl donor substrate/product adenosine triphosphate (ATP)/adenosine diphosphate (ADP). As expected, the quaternary, tertiary, and secondary structures of TcpIPK closely resembled those of IPKs published previously, and kinetic analysis of a novel alkyl-P substrate highlighted the potentially dramatic effects of altering the core scaffold of the natural substrate. Even more interesting, though, was the discovery of a trend correlating the position of two α helices in the active site with the magnitude of an IPK homolog's reaction rate for the natural reaction. Overall, the current structures of TcpIPK highlight the importance of continued structural analysis of the IPKs to better understand and optimize their activity with both natural and non-natural substrates.

N-Aryl iminochromenes inhibit cyclooxygenase enzymes via π-π stacking interactions and present a novel class of anti-inflammatory drugs.

Cyclooxygenase enzymes (COX1/2) have been widely studied and noted for their role in the biosynthesis of inflammation-induced proteins, prostaglandins and thromboxane. Multiple anti-inflammatory drugs have been developed to target these two enzymes, but most of them appeared to have notable adverse effects, especially on the cardiovascular system and lower gastrointestinal tract, suggesting an urgent need for new potent anti-inflammatory drugs. In this study, we screened twenty-two previously synthesized N-aryl iminochromenes (NAIs) for their anti-inflammatory activity by performing COX-1/2 inhibitory assays. Five compounds (1, 10, 14, 15, and 20) that gave the best in vitro anti-inflammatory results were subjected to an in vivo anti-inflammatory assay using the formalin-induced hind rat paw oedema method, followed by in silico studies using indomethacin and celecoxib as standard drugs. Among them, compound 10 stood out as the best candidate, and the percentage reduction in paw oedema at the dose of 20 mg kg-1 body weight was found to be substantially higher with compound 10 than that with indomethacin. This is mostly due to the excellent suitability of the chromene-phenyl scaffold with a highly concentrated area of aromatic residues, which produced good π-π stacking interactions. Taken together, this study strongly suggests compound 10 as a potential candidate for anti-inflammatory drug research.