DOI: 10.1002/cbic.201000537 Structural Analysis of CYP101C1 from Novosphingobium aromaticivorans DSM12444 Ming Ma , [a] Stephen G. Bell ,* [b] Wen Yang, [c] Yiming Hao, [c] Nicholas H. Rees, [b] Mark Bartlam, [c] Weihong Zhou,* [c] Luet-Lok Wong, [b] and Zihe Rao [a, c] Introduction Cytochrome P450 (CYP) enzymes constitute a superfamily of heme-containing monooxygenases, which, among other activi- ties, are able to insert an oxygen atom from dioxygen into chemically inert carbon hydrogen bonds with high regio- and stereoselectivity. [1, 2] They are of great interest due to their myriad physiological roles and potential application in the bio- catalytic synthesis of fine chemicals under mild conditions. [3–5] Dioxygen activation requires two electrons that are usually de- rived from NAD(P)H and delivered to the CYP enzymes by elec- tron transfer proteins. [6] Numerous CYP enzymes with poten- tially interesting and desirable activities have been discovered but many of these are orphaned with no electron transfer part- ners located nearby in the genome sequence, and their activi- ties are often compromised in hybrid electron transfer sys- tems. [7–11] The electron transfer proteins can also have addition- al roles, for example, putidaredoxin (Pdx) has an effector role in CYP101A1 (P450cam) activity. [12–14] Therefore, the identifica- tion and characterisation of functional CYP electron transfer chains are imperative in order to optimise the catalytic activity. We have reported the heterologous production and purifica- tion of the majority of the sixteen P450 enzymes from Novo- sphingobium aromaticivorans DSM12444. [15, 16] Potential sub- strates for ten of these enzymes have been identified and span a broad range of organic molecules including terpenoids, linear alkanes and polyaromatic hydrocarbons. [16] The CYP en- zymes of this bacterium are presumably important in enabling it to survive in the oligotrophic environments where it is found. [17, 18] A class I electron transfer system, consisting of a flavin-dependent ferredoxin reductase, ArR, and a [2Fe–2S] fer- redoxin, Arx, has been identified that is able to reconstitute the monooxygenase activity of at least five of these enzymes (CYP101B1, CYP101C1, CYP101D1, CYP101D2 and CYP111A2). [11, 15, 16] Whole-cell systems capable of product for- mation on the gram-per-litre scale in shake flasks have also been constructed. [15] CYP101D1 and CYP101D2 both bind and oxidise camphor (to yield  98% 5-exo-hydroxycamphor) while CYP101C1 and CYP101B1 are able to bind and oxidise b- ionone. Unusually, for bacterial CYP enzymes, the Arx oxidation kinetics of CYP101C1 with b-ionone as substrate shows posi- tive cooperativity. [11] Here, we report the structures of CYP101C1 with b-ionone or hexanediol bound. Both structures have open conforma- tions with an access channel in a similar position to those found in the recently solved structures of CYP101A1 and CYP101D2. [19, 20] In common with CYP101D1 and CYP101D2, the proximal face of CYP101C1 has an overall positive electrostatic potential that can interact with a negatively charged area on Arx. [11] However, compared to CYP101D1 and CYP101D2 there CYP101C1 from Novosphingobium aromaticivorans DSM12444 is a homologue of CYP101D1 and CYP101D2 enzymes from the same bacterium and CYP101A1 from Pseudomonas putida. CYP101C1 does not bind camphor but is capable of binding and hydroxylating ionone derivatives including a- and b- ionone and b-damascone. The activity of CYP101C1 was high- est with b-damascone (k cat = 86 s 1 ) but a-ionone oxidation was the most regioselective (98 % at C3). The crystal structures of hexane-2,5-diol- and b-ionone-bound CYP101C1 have been solved; both have open conformations and the hexanediol- bound form has a clear access channel from the heme to the bulk solvent. The entrance of this channel is blocked when b- ionone binds to the enzyme. The heme moiety of CYP101C1 is in a significantly different environment compared to the other structurally characterised CYP101 enzymes. The likely ferredox- in binding site on the proximal face of CYP101C1 has a differ- ent topology but a similar overall positive charge compared to CYP101D1 and CYP101D2, all of which accept electrons from the ArR/Arx class I electron transfer system. [a] Dr. M. Ma, + Prof. Z. Rao National Laboratory of Macromolecules, Institute of Biophysics Chinese Academy of Science, Beijing 100101 (China) [b] Dr. S. G. Bell , + Dr. N. H. Rees, Dr. L.-L. Wong Department of Chemistry, University of Oxford Inorganic Chemistry Laboratory, South Parks Road Oxford OX1 3QR (UK) Fax: (+ 44) 1865-272690 E-mail : stephen.bell@chem.ox.ac.uk [c] W. Yang, Y. Hao, Prof. M. Bartlam, Dr. W. Zhou, Prof. Z. Rao Tianjin Key Laboratory of Protein Science, College of Life Sciences Nankai University, Tianjin 300071 (China) Fax: (+ 86) 22-23502351 E-mail : zhouwh@nankai.edu.cn [ + ] These authors contributed equally to the work. Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000537. 88  2011 Wiley-VCH Verlag GmbH& Co. KGaA, Weinheim ChemBioChem 2011, 12, 88 – 99