虚拟专刊 | Virtual Issue on Enzyme Regulation & Function

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An Argonaute from Thermus parvatiensis exhibits endonuclease activity mediated by 5′ chemically modified DNA guides

Yingying Sun, Xiang Guo, Hui Lu, Liuqing Chen, Fei Huang, Qian Liu, Yan Feng.

Acta Biochim Biophys Sin 2022, 54: 686-695.

Argonaute (Ago) proteins are a family of nucleic acid-guided proteins sharing a conserved domain architecture for the N-terminal, PAZ, MID, and PIWI domains. Extensive biochemical studies have revealed that recognition of guides with different 5′ groups by Ago is important for biocatalysis.

• We identified an Ago from the thermophilic Thermus parvatiensis (TpsAgo) and analyzed the regulatory effect of 5′-modified guides on TpsAgo cleavage activity.

• TpsAgo can utilize various 5′-modified DNA guides for catalysis, including 5′-NH2C6, 5′-Biotin, 5′-FAM and 5′-SHC6 guides.

• TpsAgo cleaves single-stranded DNA and RNA targets at 65°C–90°C, which is mediated by a 5′ hydroxyl or phosphate DNA guide, and double-stranded DNA at over 80°C. 

Translesion synthesis of apurinic/apyrimidic siteanalogues by Y-family DNA polymerase Dbh from Sulfolobus acidocaldarius

Weiwei Wang, Huan Zhou, Li Peng, Feng Yu, Qin Xu, Qisheng Wang, Jianhua He, Xipeng Liu.

Acta Biochim Biophys Sin 2022, 54: 202-214.

Apurinic/apyrimidic (AP) sites are severe DNA damages and strongly block DNA extension by major DNA polymerases. Y-family DNA polymerases possess a strong ability to bypass AP sites and continue the DNA synthesis reaction, which is called translesion synthesis (TLS) activity.

• The molecular structure and atomic composition but also the number and position of AP site analogues determine the TLS efficiency of Dbh.

• Increasing the spacer length decreases TLS activity. The position of the AP site analogues is also an important factor for TLS.

• When the spacer is opposite to the first incorporated dNTPs, the TLS efficiency is the lowest, suggesting that AP sites are largely harmful for the formation of hydrogen bonds.

Parthenolide reveals an allosteric mode to inhibit the deISGylation activity of SARS-CoV‑2 papain-like protease

Zhihui Zou, Huizhuang Shan, Demeng Sun, Li Xia, Yulong Shi, Jiahui Wan, Aiwu Zhou, Yunzhao Wu, Hanzhang Xu, Hu Lei, Zhijian Xu, Yingli Wu.

Acta Biochim Biophys Sin 2022, 54: 1133-1139.

The coronavirus papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for viral polypeptide cleavage and the deISGylation of interferon-stimulated gene 15 (ISG15), which enable it to participate in virus replication and host innate immune pathways. Therefore, PLpro is considered an attractive antiviral drug target.

• Parthenolide inhibits the deISGylation activity of SARS-CoV‑2 PLpro.

• Parthenolide covalently modifies Cys-191 or Cys-194 of PLpro.

• Mutations of Cys-191 or/and Cys-194 markedly reduce PLpro-induced cleavage of proISG15.

Crystal structure of the phosphorylated Arabidopsis MKK5 reveals activation mechanism of MAPK kinases

Chao-Jun Pei, Qing-Xia He, Zhipu Luo, Hongwei Yao, Zhi-Xin Wang, Jia-Wei Wu.

Acta Biochim Biophys Sin 2022, 54: 1159-1170.

MKKs are central kinases of the three-tier MAPK cascades, and how MKKs bind and activate their cognate substrates is important for precise signal transmission. Here, we show that Arabidopsis MKK5 is the effective upstream kinase of AtMPK6, and the N-terminal KIM of AtMKK5 is indispensable for substrate recognition. The crystal structure of phosphorylated AtMKK5 unravels the activation mechanism of MKKs, as well as the molecular basis of substrate specificity.

• AtMKK5 effectively catalyzes the dual phosphorylation of AtMPK6.

• AtMKK5 contains a conserved KIM essential for specific substrate recognition.

• Structure of AtMKK5 kinase domain revealed that phosphorylation at both Thr and Ser residues within the activation segment is required to establish the active conformation of MKKs.

Aromatic disulfides as potential inhibitors against interaction between deaminase APOBEC3G and HIV infectivity factor

Xiaoxuan Yan, Chao Chen, Chunxi Wang, Wenxian Lan, Jianguo Wang, Chunyang Cao.

Acta Biochim Biophys Sin 2022, 54: 725-735.

APOBEC3G (A3G) displays activities against retrovirus and retrotransposon by inhibition of virus infectivity factor (Vif)-deficient HIV-1 replication. The interaction between A3G N-terminal domain and Vif directs the cellular Cullin 5 E3-ubiquitin ligase complex to ubiquitinate A3G, and leads to A3G proteasomal degradation, which is a potential target for anti-HIV drug. 

• We screened two series of small molecules containing carbamyl sulfamide bond or disulfide bond as bridges of two different aromatic rings. 

• Five asymmetrical disulfides were successfully identified against interaction between A3G and Vif with the IC50 values close to or smaller than 1 μM, especially, not through covalently binding with A3G or Vif. 

• They restore the A3G expression in the presence of Vif by inhibiting Vif-induced A3G ubiquitination and degradation. 

PHD3 inhibits cell proliferation through hydroxylation of PAX2 at proline 9

Jie Lun, Yuxin Wang, Qiang Gao, Yu Wang, Hongwei Zhang, Jing Fang.

Acta Biochim Biophys Sin 2022, 54: 708-715.

The oncoprotein transcription factor paired box 2 (PAX2) is aberrantly expressed in cancers, but the underlying mechanism remains elusive. Prolyl hydroxylase 3 (PHD3) hydroxylates the proline residue of HIFα, mediating HIFα degradation. The von Hippel-Lindau protein (pVHL) is an E3 ligase which mediates ubiquitination and degradation of hydroxylated HIFα. 

• PHD3 hydroxylates PAX2 at proline 9, which is required for pVHL to mediate PAX2 ubiquitination and degradation. 

• pVHL does not bind with PAX2(P9A) and cannot enhance its ubiquitination and degradation.

• PAX2 promotes cell proliferation, which is suppressed by co-expression of PHD3 but not by hydroxylase-deficient PHD3(H196A). 

• PHD3 inhibits PAX2-induced, but not PAX2(P9A)-induced proliferation of COS-7 cells. 

Data-driven strategies for the computational design of enzyme thermal stability: trends, perspectives, and prospects

Zhixin Dou, Yuqing Sun, Xukai Jiang, Xiuyun Wu, Yingjie Li, Bin Gong, Lushan Wang.

Acta Biochim Biophys Sin 2023, 55: 343-355.

Thermal stability is one of the most important properties of enzymes which receives much attention in enzyme engineering. Here, we review some widely adopted datasets and data-driven approaches for the thermal stability design of enzymes. In addition, we discuss a series of existing challenges while applying machine learning in enzyme thermostability design.

• Datasets related to enzyme thermostability come from two different sources, manual collection and predictive modeling.

• Choosing the efficient data representation methods for sequence and structure data is an important step in embedding biological knowledge into machine learning models.

• The utilization of published datasets and models still has a series of problems and challenges in terms of ease of use, standardization and interpretability.

Computational investigations on target-site searching and recognition mechanisms by thymine DNA glycosylase during DNA repair process

Lingyan Wang, Kaiyuan Song, Jin Yu, Lin-Tai Da.

Acta Biochim Biophys Sin 2022, 54: 796-806.

DNA glycosylase, as one member of DNA repair machineries, plays an essential role in correcting mismatched/damaged DNA nucleotides by cleaving the N-glycosidic bond between the sugar and target nucleobase through the base excision repair (BER) pathways. 

• We reveal atomic-level structural dynamics of TDG and pinpoint the key structural elements responsible for regulating the translocation of TDG along DNA. 

• TDG adopts a base-flipping mechanism to extrude the mispaired nucleobase into the enzyme active-site.  

• The molecular mechanism of product release dynamics after catalysis is also summarized.