title: Crystal structure of PnpCD, a two-subunit hydroquinone 1,2-dioxygenase, reveals a novel structural class of Fe2+-dependent dioxygenases.
abstract: Aerobic microorganisms have evolved a variety of pathways to degrade aromatic and heterocyclic compounds. However, only several classes of oxygenolytic fission reaction have been identified for the critical ring cleavage dioxygenases. Among them, the most well studied dioxygenases proceed via catecholic intermediates, followed by noncatecholic hydroxy-substituted aromatic carboxylic acids. Therefore, the recently reported hydroquinone 1,2-dioxygenases add to the diversity of ring cleavage reactions. Two-subunit hydroquinone 1,2-dioxygenase PnpCD, the key enzyme in the hydroquinone pathway of para-nitrophenol degradation, catalyzes the ring cleavage of hydroquinone to γ-hydroxymuconic semialdehyde. Here, we report three PnpCD structures, named apo-PnpCD, PnpCD-Fe(3+), and PnpCD-Cd(2+)-HBN (substrate analog hydroxyenzonitrile), respectively. Structural analysis showed that both the PnpC and the C-terminal domains of PnpD comprise a conserved cupin fold, whereas PnpC cannot form a competent metal binding pocket as can PnpD cupin. Four residues of PnpD (His-256, Asn-258, Glu-262, and His-303) were observed to coordinate the iron ion. The Asn-258 coordination is particularly interesting because this coordinating residue has never been observed in the homologous cupin structures of PnpCD. Asn-258 is proposed to play a pivotal role in binding the iron prior to the enzymatic reaction, but it might lose coordination to the iron when the reaction begins. PnpD also consists of an intriguing N-terminal domain that might have functions other than nucleic acid binding in its structural homologs. In summary, PnpCD has no apparent evolutionary relationship with other iron-dependent dioxygenases and therefore defines a new structural class. The study of PnpCD might add to the understanding of the ring cleavage of dioxygenases.
authors: From the State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, Shandong 250100.
authors: Shiheng
authors: S
authors: Liu
authors: From the State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, Shandong 250100.
authors: Tiantian
authors: T
authors: Su
authors: From the State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, Shandong 250100.
authors: Cong
authors: C
authors: Zhang
authors: the Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071.
authors: Wen-Mao
authors: WM
authors: Zhang
authors: From the State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, Shandong 250100.
authors: Deyu
authors: D
authors: Zhu
authors: the College of Food Science and Engineering, Qilu University of Technology, Jinan, Shandong 250353, and.
authors: Jing
authors: J
authors: Su
authors: From the State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, Shandong 250100.
authors: Tiandi
authors: T
authors: Wei
authors: From the State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, Shandong 250100.
authors: Kang
authors: K
authors: Wang
authors: From the State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, Shandong 250100.
authors: Yan
authors: Y
authors: Huang
authors: the Rizhao Center for Diseases Prevention and Control, Rizhao Health Bureau, Rizhao, Shandong 276826, China.
authors: Liming
authors: L
authors: Guo
authors: From the State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, Shandong 250100.
authors: Sujuan
authors: S
authors: Xu
authors: the Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, the State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, ningyi.zhou@sjtu.edu.cn.
authors: Ning-Yi
authors: NY
authors: Zhou
authors: From the State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, Shandong 250100, lcgu@sdu.edu.cn.
authors: Lichuan
authors: L
authors: Gu
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D049308:Dioxygenases / Q000737:chemistry*; D006873:Hydroquinones / Q000737:chemistry*; D007477:Ions; D007501:Iron / Q000737:chemistry; D008660:Metabolism; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009570:Nitriles / Q000737:chemistry; D009596:Nitrophenols / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D006873:Hydroquinones; D007477:Ions; D008670:Metals; D009570:Nitriles; D009596:Nitrophenols; C014356:benzonitrile; D007501:Iron; D049308:Dioxygenases; D010100:Oxygen; C031927:hydroquinone; C024836:4-nitrophenol
keywords: biodegradation; cupin; dioxygenase; enzyme mechanism; metal coordination; metalloenzyme; ring cleavage; structure-function