Structure and Action of the Myxobacterial Chondrochloren Halogenase CndH: A New Variant of FAD-dependent Halogenases Stefan Buedenbender 1 , Shwan Rachid 2 , Rolf Müller 2 and Georg E. Schulz 1 ⁎ 1 Institut für Organische Chemie und Biochemie, Albert-Ludwigs- Universität, Albertstr. 21, D-79104 Freiburg im Breisgau, Germany 2 Pharmazeutische Biotechnologie, Universität des Saarlandes, P.O. Box 151150, D-66041 Saarbrücken, Germany Received 15 August 2008; received in revised form 11 October 2008; accepted 21 October 2008 Available online 30 October 2008 The crystal structure of the FAD-dependent chondrochloren halogenase CndH has been established at 2.1 Å resolution. The enzyme contains the characteristic FAD-binding scaffold of the glutathione reductase super- family. Except for its C-terminal domain, the chainfold of CndH is virtually identical with those of FAD-dependent aromatic hydroxylases. When compared to the structurally known FAD-dependent halogenases PrnA and RebH, CndH lacks a 45 residue segment near position 100 and deviates in the C-terminal domain. Both variations are near the active center and appear to reflect substrate differences. Whereas PrnA and RebH modify free tryptophan, CndH halogenates the tyrosyl group of a chondrochloren precursor that is most likely bound to a carrier protein. In contrast to PrnA and RebH, which enclose their small substrate completely, CndH has a large non-polar surface patch that may accommodate the putative carrier. Apart from the substrate binding site, the active center of CndH corresponds to those of PrnA and RebH. At the halogenation site, CndH has the characteristic lysine (Lys76) but lacks the required base Glu346 (PrnA). This base may be supplied by a residue of its C-terminal domain or by the carrier. These differences were corroborated by an overall sequence comparison between the known FAD-dependent halogenases, which revealed a split into a PrnA-RebH group and a CndH group. The two functionally established members of the CndH group use carrier-bound substrates, whereas three members of PrnA-RebH group are known to accept a free amino acid. Given the structural and functional distinction, we classify CndH as a new variant B of the FAD-dependent halogenases, adding a new feature to the structurally established variant A enzymes PrnA and RebH. © 2008 Elsevier Ltd. All rights reserved. Edited by R. Huber Keywords: halogenated antibiotics; crystal structure; FAD-dependent halogenases; Myxobacteria; X-ray diffraction Introduction Halogenated natural products are widespread, and more than 4500 compounds are known. 1 Halogenation is particularly abundant in antibio- tics, where this modification usually increases the activity. 2 Most halogenated molecules are of terrestrial origin and contain chlorine. In contrast, marine organisms use predominantly bromine. 3 Fluorinated biomolecules are rare. 3-5 The best known iodine derivative is the hormone thyroxine. The halogen-introducing enzymes are classified as *Corresponding author. E-mail address: georg.schulz@ocbc.uni-freiburg.de. Abbreviations used: CmdE, chondramide halogenase from Chondromyces crocatus; CndH, chondrochloren halogenase from Chondromyces crocatus; pHBH, para-hydroxybenzoate 3-hydroxylase from Pseudomonas fluorescens; PltA, pyoluteorin halogenase from Pseudomonas fluorescens; PrnA, pyrrolnitrin halogenase from Pseudomonas fluorescens; Puthal, putative halogenase from Shewanella frigidimarina; PyrH, pyrroindomycin halogenase from Streptomyces rugosporus; RebH, rebeccamycin halogenase from Lechevalieria aerocolonigenes. doi:10.1016/j.jmb.2008.10.057 J. Mol. Biol. (2009) 385, 520–530 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.