ABSTRACT
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.
Subject(s)
Adenosine Triphosphate , Catalytic Domain , Thermococcus , Substrate Specificity , Thermococcus/enzymology , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/chemistry , Crystallography, X-Ray , Models, Molecular , Protein Binding , Kinetics , Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Archaeal Proteins/genetics , Hemiterpenes/metabolism , Hemiterpenes/chemistry , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/genetics , Protein Conformation, alpha-Helical , Adenosine Diphosphate/metabolism , Adenosine Diphosphate/chemistry , Cloning, Molecular , Gene Expression , Protein Conformation, beta-Strand , Amino Acid Sequence , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli/enzymology , Protein KinasesABSTRACT
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.
