Characterization of JadH as an FAD- and NAD(P)H-dependent bifunctional hydroxylase/dehydrase in jadomycin biosynthesis.

As a major class of aromatic polyketide natural products, the angucyclines display significant diversity in structure and bioactivity. Like most other bacterial aromatic polyketides, angucyclines are derived from polyketide chains assembled by the minimal type II polyketide synthases, which consist of a ketosynthase a and b heterodimer complex and a cognate acyl carrier protein. The polyketide chains are then modified by accessory enzymes (ketoreductase and cyclase/aromatase) to generate the key angucycline intermediates, UWM6 (1) or its analogues. Subsequent modification steps catalyzed by tailoring enzymes afford the hundreds of structurally diverse angucyclines that have been identified. A large number of different kinds of enzymes are involved in the tailoring steps of angucyclines. Investigation of these enzymes will help us to understand the mechanism of biosynthetic assembly of these molecules, and this knowledge is required for combinatorial engineering to produce novel angucyclines. Among all the tailoring steps, C12 monooxygenation is remarkable in that it takes place in all angucycline biosynthetic pathways, and investigation of the enzyme catalyzing this reaction should shed significant light on the biosyntheses of the whole angucycline family. The first characterized enzyme catalyzing angucycline C12 monooxygenation is JadH from jadomycin (JD) pathway. JD refers to a series of angucycline molecules with a unique pentacyclic benz[b]oxazolophenanthridine skeleton. They exhibit good bioactivity against Gram-positive bacteria, including the methicillin-resistant Staphylococcus aureus. Recently, JD B (2 ; Scheme 1) was shown to inhibit the activity of Aurora-B kinase. The jad gene cluster was cloned from Streptomyces venezuelae ISP5230 in the early 1990s, and the JD biosynthetic pathway has been thoroughly analyzed. JadH was proposed to catalyze the C12 monooxygenation and 4a,12b-dehydration from mainly in vivo investigations. It was also shown to convert 2,3-dehydro-UWM6 (3) to dehydrorabelomycin (4) and 1 to rabelomycin (5) in vitro (Scheme 2). Studies on the JadH homologues, (for example, PgaE (55.9% identity) from the gaudimycin A (6), CabE (55.1% identity) from the gaudimycin B (7), OvmOI (54.4% identity) from the oviedomycin (8), and GilOI (40.1% identity) from the gilvocarcin V (9) gene clusters, 18] in other angucycline pathways also support the C12 monooxygenation function of JadH (Scheme 1). However, two questions remain when comparing the function of JadH with its homologues. First, the biosynthesis of 9 should evoke a hydroquinone instead of a quinone intermediate, which suggests that C12 hydroxylation is the real reaction catalyzed by JadH homologues in the 9 biosynthesis. It is also consistent with the similarities between JadH homologues and typical FAD-dependent aromatic hydroxylases in primary, secondary and tertiary structures. Second, non-enzymatic 4a,12b-dehydration has been observed previously in the spontaneous conversion from 1 to 5 (Scheme 2), and the 4a-hydroxyl groups are kept in some angucyclines (for example, 6, 7 and simocyclinone D8 (10)), hence raising the question whether the 4a,12b-dehydration is catalyzed by the JadH homologues or whether it is just a non-enzymatic conversion. These intriguing questions motivated us to further characterize the function of angucycline C12 monooxygenation enzymes, such as JadH. The N-His-tagged JadH (JadH-N-His) was expressed and purified as described previously. To overexpress native JadH in Escherichia coli, plasmid pET23b-jadH was first introduced into E. coli BL21. However, no JadH expression was detected although different culture temperatures and IPTG concentrations were tested. We noticed that several codons (CGG and CCC) seldom used in E. coli appeared frequently in the jadH gene. The plasmid was then transformed into the E. coli host Rosetta gami B LysS, which harbors plasmids for E. coli-rare codons. Although it was not very efficient, native JadH was expressed as soluble protein in the Rosetta host. About 0.4 mg of colorless native JadH was purified from the cell extracts of 1 L cultured cells. Purified native JadH and JadH-N-His were examined on 10% SDS-PAGE. Both of them migrated as a single band with a molecular mass consistent with the predicted sizes (57 kDa for native JadH (Figure S1 in the Supporting Information) and 61 kDa for JadH-N-His (Figure 1)). JadH was proposed to be an FAD-dependent enzyme based on it containing a typical Rossmann fold dinucleotide-binding motif (GXGXXG/A), which is responsible for binding the adenosine moiety of FAD, and a GD motif interacting with the flavin moiety of FAD (Figure S2). Purified JadH-N-His is yellow and its UV/visible spectrum was consistent with the presence of a flavin cofactor with absorbance maxima at 366 and 450 nm (Figure S3). After JadH-N-His was denatured by acidification (5 mL of 1m HCl was added to 50 mL of 10 mm [a] Dr. Y. Chen , Dr. K. Fan , Y. He, Dr. X. Xu, Dr. Y. Peng, T. Yu, C. Jia, Prof. Dr. K. Yang State Key Laboratory of Microbial Resources, Institute of Microbiology Chinese Academy of Sciences Beijing, 100101 (China) Fax: (+86)10-64807459 E-mail : [email protected] [b] Dr. Y. Chen + Present address: Division of Pharmaceutical Sciences, University of Wisconsin–Madison 777 Highland Avenue, Madison, WI 53705 78 (USA) [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201000178.