Book Description

Biologically active halogenated compounds are widespread, and halogen atoms play a crucial role in their biological activity. Flavin-dependent halogenases (FDHs) are halogenating enzymes from bacteria and fungi that regioselectively form aryl halides under mild conditions in an aqueous solution. Enzymatic halogenation is a greener alternative for aryl halide formation compared to conventional halogen chemistry. The regioselectivity of FDHs overrides electronic directing effects. Thus, FDHs have the potential to be useful as synthetic biocatalysts, especially in pharmaceutical and agrochemical industries. We have expressed and purified AbeH, a gene product from a bacterial bisindole alkaloid biosynthetic gene cluster and demonstrated that it is a novel regioselective FDH with the ability to convert FADH2, O2, Cl- and Tryptophan (Trp) to FAD, H2O, and 5-Cl-Trp. AbeH can also brominate Trp and halogenate several other aromatic compounds. We have solved the 1.86 Å X-ray crystal structure of AbeH with bound FAD and the structure of Apo-AbeH at 1.89 Å. AbeH structures shows conserved fold of FDHs, and FAD bound AbeH structure revealed a conserved FAD-binding site. Attempts to solve crystal structures of the AbeH-FAD-Trp ternary complex or the AbeH-Trp binary complex were unsuccessful. Despite efforts at cocrystallization and soaking, thirteen data sets were collected, but none had electron density for Trp. An X-ray structure solved previously by Dr. Kazi Lingkon of the Trp-6-halogenase BorH with FAD and Trp also shows no ternary complex formation. Instead, ligands are present on different molecules in the asymmetric unit, never together bound to the same chain. We also noted that FAD inhibited the in vitro chlorination of Trp by AbeH, a phenomenon also observed for BorH. These observations from crystallographic analysis, along with FAD-induced halogenated product inhibition, led us to hypothesize that AbeH and BorH have some allosteric communication between the ~10 Å distant FAD and Trp binding sites leading to negative cooperativity. To investigate this potential allosteric regulation binding of FAD and Trp to both enzymes has been analyzed with isothermal titration calorimetry. Both AbeH and BorH can form binary complexes with either FAD or Trp, but with a marked difference in their affinity for FAD versus Trp, where FAD-binding is preferred in both enzymes. Titration of one ligand into a binary complex with the other ligand showed no evidence of ternary complex formation for BorH, suggesting that the first ligand-bound has maximal negative cooperativity for second ligand binding. For AbeH, Trp cannot bind to the AbeH/FAD complex, but FAD can bind to AbeH/Trp saturated complex. Structural analysis suggests that the architecture of the Trp binding site may play a crucial role in allosteric regulation and explain the different ITC results of AbeH and BorH for detection of FAD-binding to the binary complexes with Trp.