Crystal structure of AlpK: An essential monooxygenase involved in the biosynthesis of kinamycin

https://doi.org/10.1016/j.bbrc.2019.01.077Get rights and content

Highlights

  • The structure of AlpK in complex with FAD from Streptomyces ambofaciens.

  • The FAD molecule adopts an “open” conformation.

  • Two putative substrate binding sites are predicted.

Abstract

AlpK is an essential monooxygenase involved in the biosynthesis of kinamycin. It catalyzes the C5-hyfroxylattion of the crucial benzo[b]-fluorence intermediate in kinamycin synthesis. However, the structure and mechanism of AlpK is unclear. Here, we report the first structure of AlpK in complex with FAD. Our structure sheds light on the catalytic mechanism of AlpK.

Introduction

Kinamycin is an atypical angucycline which is produced by several Streptomyces spp. [1]. Kinamycin is biosynthesized by a type II polyketide synthase (PKS) gene cluster known as angucycline-like polyketide (alp) [[2], [3], [4]]. The biosynthesis of angucyclines start at the assembly of a linear polyketide chain by Claisen condensation of short chain acyl-CoA (usually acetyl-CoA or its derivative malonyl-CoA). The nascent polyketide chain is then modified by ketoreductases, cyclases and aromatases to generate biosynthetic intermediates with angucycline-type skeleton [5,6]. After that, various tailoring enzymes catalyze the subsequent modification steps, which are the major cause of the structural diversion of angucyclines [5,7]. FAD-dependent monooxygenases are a major family of angucycline tailoring enzymes. The biosynthetic gene clusters of angucyclines usually contain at least two conserved FAD-dependent monooxygenases. One is proposed to catalyze the 2,3-dehydration reaction and the other catalyze hydroxylation and dehydration reactions at various positions [5,6]. However, the biosynthetic gene cluster of kinamycin, as well as similar gene clusters of lomaiviticin and fluostatin, has up to five FAD-dependent monooxygenases, while the catalytic functions are still unclear for most of these extra FAD-dependent monooxygenases [[8], [9], [10], [11], [12], [13], [14]]. One of them, AlpK, is involved in the ring opening reaction of the key intermediate dehydrorabelomycin (DHR). The ring opening reaction of DHR is catalyzed by AlpJ, a unique family of oxygenase, in which the six-member B ring of DHR is opened and rearranges to a contracted five-member ring of the unstable product, the benzo[b]fluorene intermediate [15]. It was proposed that AlpK catalyzed the C5-hydroxylation reaction of the benzo[b]-fluorene intermediate. And it was also reported that AlpK could supply necessary FADH2 for the AlpJ catalyzed reaction [15].

AlpK is a new catalytic entity in the pHBH (p-hydroxybenzoic acid hydroxylase) family enzymes [15]. The pHBH family protein contains three domains: the FAD-binding domain, the middle domain and the C-terminal domain [[16], [17], [18], [19], [20], [21], [22], [23]]. The FAD-binding domain provides the binding sites both for the cofactor FAD and the co-substrate NADPH. The middle domain possesses the substrate binding site and regulates the catalytic states. The function of the C-terminal domain is still unclear. Particularly, the isoalloxazine ring of FAD is the critical catalytic core in FAD-dependent enzyme. Three conformations of the FAD were observed in the structures of pHBH family proteins, known as “in” [17,18,20], “open” [16] and “out” [21,23]. The “open” conformation is considered as an essential initial step during which the FAD is exposure to the solvent and ready for the binding of substrate [[16], [17], [18], [19]]. After that, the substrate enter the tunnel and localizes at the active site [16,17,19]. The conformation of FAD will be changed to “in” after substrate binding. Then, it will be reduced by NADPH and changed to the “out” conformation [16,17,19,21,23]. In this process, the structure of the FAD binding domain is also changed, and was named as “open”, “in” and “out”, accordingly [16,17,19,21,23].

Although AlpK performs important function in kinamycin synthesis, the mechanism of it is still unclear. Here we report the crystal structure of AlpK in complex with FAD with an “open” conformation. The structure analysis shows two putative substrate binding sites for the substrate. These results provide insight into the function and catalytic mechanism of AlpK.

Section snippets

Clone, expression and purification

The AlpK gene was cloned into a modified pRSF-Duet plasmid with 6 × His at its N-terminal. Protein expression was induced with 0.2 mM isopropyl-d-thiogalactopyranoside at 16 °C in E.coli BL21 (DE3) for 18 h. The bacteria cell was harvested and resuspended in lysis buffer (20 mM HEPES pH 7.4, 200 mM NaCl, 5% glycerol). The cell was then sonicated in ice-bathing condition followed by high-speed centigrade at 16,000 rpm. The proteins in the supernatant were purified by nickle affinity

Overall structure of AlpK

The crystal structure of AlpK in complex with FAD was determined at a resolution of 2.89 Å (Table 1). AlpK was crystalized in a P6122 space group, with one molecule in the asymmetric unit. Similar to other pHBH family members, the structure of AlpK is composed of the FAD binding domain (residues 1–173 and 263–377), the middle domain (residues 174–262) and the C-terminal domain (residues 390–491) (Fig. 1A). The FAD binding domain of AlpK serves as the catalytic domain of the enzyme. Comprising 8

Discussion

In this paper, we present the crystal structure of AlpK with FAD in an “open” conformation. Our structure showed that the loop1 (residues 185–196) show a significant conformation change compared with PgaE. The loop2 (residues 286–289) may assist to stabilize the FAD “open” conformation through hydrophobic interaction by Pro286. In the “open” conformational AlpK, a tunnel exists between the FAD binding domain and middle domain. This tunnel lead to the exposure to solvent and enable the substrate

Contributions

W. Wang performed experiments and write the manuscript. H. Li and J. Li collected and processed the diffraction data. J. Li determined the structure. J. Li and W. Wang refined the structure. K. Fan and Y. Liu revised the manuscript. All authors reviewed the results and approved the final version of the manuscript.

Acknowledgements

We Thank Dr. Zhonghui Tang and Dr. Lixin Zhu for helpful discussion. We thank the scientists in Shanghai Synchrotron Radiation Facility SSRF BL17U for assistance using the facility during data collection. This work was supported by National Natural Science Foundation of China (31530015, 31500609).

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