Research progress: Selective oxidation of aliphatic C–H bonds in alkylphenols by a “chemomimetic” biocatalytic system

[2017.6.12] We collaborated with the Enzyme Engineering Group (Prof. Shengying Li) in a PNAS paper to analyze the mechanism of the selective alkylphenol side chain oxidation by a new P450 monooxygenase.

See the paper:
Lei Du†, Sheng Dong†, Xingwang Zhang, Chengying Jiang, Jingfei Chen, Lishan Yao, Xiao Wang, Xiaobo Wan, Xi Liu, Xinquan Wang, Shaohua Huang, Qiu Cui, Yingang Feng*, Shuang-Jiang Liu*, and Shengying Li* (2017) Selective oxidation of aliphatic C-H bonds in alkylphenols by a chemomimetic biocatalytic system. Proc. Natl. Acad. Sci. USA Inpress. [PMID: 28607077] [Full text(Publisher website)]

(The following contents are copied from the news in the website of the Institute.)

Alkylphenols are among the most important synthetic precursors for manufacturing a great variety of chemical products including detergents, polymers, lubricants, antioxidants, emulsifiers, pesticides, and pharmaceuticals. Alkylphenols are also priority environmental pollutants because they are toxic, xenoestrogenic, or carcinogenic to wildlife and humans. The selective oxidation of the aliphatic C-H bonds in alkylphenols serves significant roles not only in biological degradation of these environmentally hazardous compounds, but also in generation of functionalized intermediates for synthesizing more downstream products. In particular, some oxidized alkylphenols are direct structural motifs in drugs (e.g., Metoprolol) and bioactive ingredients (e.g., Gastrodin, Picein and Salidroside) of medicinal plants.

Recently, the Enzyme Engineering Group directed by Professor Shengying Li at Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, together with the research team led by Professor Shuang-Jiang Liu at Institute of Microbiology, Chinese Academy of Sciences, identified a novel p-cresol biodegradation pathway encoded by the “cre” gene cluster of the Gram-positive bacterium Corynebacterium glutamicum. The catalytic activities of all seven Cre enzymes including CreCDEFGHI were completely characterized, and the whole catabolic pathway was reconstituted in vitro. Initially, p-cresol is phosphorylated to 4-methylphenyl phosphate by a novel 4-methylphenyl phosphate synthase, CreHI. Next, a unique P450 system CreJEF specifically recognizes phosphorylated intermediates and successively oxidizes the aromatic methyl group into carboxylic acid via alcohol and aldehyde intermediates. Finally, the phosphohydrolase CreD is responsible for removal of the phosphate group to afford 4-hydroxybenzyl alcohol, 4-hydroxybenzyl aldehyde and 4-hydroxybenzyl acid. In addition, the alcohol dehydrogenase CreG and the aldehyde dehydrogenase CreC are recruited to reinforce p-cresol degradation efficiency.

 

It is striking that this unique biodegradation pathway, featuring protection/direction (phosphorylation by CreHI), oxidation (by CreJEF, CreG, and CreC), and deprotection (phosphohydrolysis by CreD) steps, mimics the process of classical chemical oxidations. Such a chemomimetic biocatalytic process is extremely rare, if not unprecedented, in nature. Realizing these, the team decided to test if the general applicability of this chemomimitic system and the high selectivity of bio-oxidation could be combined, by which we would make a significant contribution to solving the problem on selective oxidation of C-H bonds in alkyphenols.

To broaden the substrate selectivity of this unique biocatalytic system, the team of Professor Shengying Li, collaborating with the team of Professor Yingang Feng at the same institute, first solved the X-ray crystal structure of the central P450 biocatalyst CreJ in complex with its substrate p-cresol. With understanding of the structural basis for substrate recognition of this unique P450 enzyme, a biocatalytic system comprising CreHI, CreJEF/CreG/CreC, and CreD, that is able to selectively oxidize a suite of p- and m-alkylated phenols, was successfully developed. The biocatalytic processes involved various types of oxidative reactions including hydroxylation, dehydrogenation, and C-C bond cleavage. In particular, absolute stereoselectivity was achieved during the hydroxylation on the benzyl position of 4-ethylphenol and 3-ethylphenol. This is extremely difficult to achieve in chemical catalysis. Further elucidation of the co-crystal structure of CreJ and p-ethylphenyl phosphate revealed the molecular mechanism for the regio- and stereoselectivity of CreJ. Through careful management of this multi-enzyme oxidation system, the oxidation levels of 4-ethylphenol could be deliberately and delicately controlled.

Furthermore, this chemomimetic biocatalytic system was found to be able to selectively oxidize not only alkylphenols, but also aliphatic alcohols. The broad substrate selectivity demonstrated its great potential in practical application. Bioinformatics analyses revealed that a large number of similar oxidation systems exist in diverse microorganisms, from which analogous oxidative biocatalysts with different substrate preference, improved oxidation selectivity, and higher catalytic efficiency could be identified in the future.

This work was supported by National Natural Science Foundation of China under the Grant NSFC 31422002, Shandong Provincial Natural Science Foundation Grant JQ201407, and the Key Frontier Project of Chinese Academy of Sciences, No. QYZDB-SSW-SMC042 (to Shengying Li), the 973 Project from Ministry of Science and Technology Grant 2012CB7211-04 (to Shuang-Jiang Liu), and the Grant NSFC 31270784 from National Natural Science Foundation of China (to Yingang Feng).

 References:

1. Lei Du, Li Ma, Feifei Qi, Xianliang Zheng, Chengying Jiang, Ailei Li, Xiaobo Wan, Shuang-Jiang Liu, and Shengying Li, Characterization of a unique pathway for 4-cresol catabolism initiated by phosphorylation in Corynebacterium glutamicum. Journal of Biological Chemistry, 2016. 291(12): p. 6583-6594.

2. Lei Du, Sheng Dong, Xingwang Zhang, Chengying Jiang, Jingfei Chen, Lishan Yao, Xiao Wang, Xiaobo Wan, Xi Liu, Xinquan Wang, Shaohua Huang, Qiu Cui, Yingang Feng, Shuang-Jiang Liu, and Shengying Li, Selective oxidation of aliphatic C-H bonds in alkylphenols by a chemomimetic biocatalytic system. Proceedings of the National Academy of Sciences of the United States of America, 2017. Published ahead of print June 12, 2017, doi:10.1073/pnas.1702317114.