title: Crystal structure of the γ-hydroxymuconic semialdehyde dehydrogenase from Pseudomonas sp. strainWBC-3, a key enzyme involved in para-Nitrophenol degradation.
abstract: BACKGROUND
  para-Nitrophenol (PNP) is a highly toxic compound with threats to mammalian health. The pnpE-encoded γ-hydroxymuconic semialdehyde dehydrogenase catalyzes the reduction of γ-hydroxymuconic semialdehyde to maleylacetate in Pseudomonas sp. strain WBC-3, playing a key role in the catabolism of PNP to Krebs cycle intermediates. However, the catalyzing mechanism by PnpE has not been well understood.
  
  
  RESULTS
  Here we report the crystal structures of the apo and NAD bound PnpE. In the PnpE-NAD complex structure, NAD is situated in a cleft of PnpE. The cofactor binding site is composed of two pockets. The adenosine and the first ribose group of NAD bind in one pocket and the nicotinamide ring in the other.
  
  
  CONCLUSIONS
  Six amino acids have interactions with the cofactor. They are C281, E247, Q210, W148, I146 and K172. Highly conserved residues C281 and E247 were identified to be critical for its catalytic activity. In addition, flexible docking studies of the enzyme-substrate system were performed to predict the interactions between PnpE and its substrate γ-hydroxymuconic semialdehyde. Amino acids that interact extensively with the substrate and stabilize the substrate in an orientation suitable for enzyme catalysis were identified. The importance of these residues for catalytic activity was confirmed by the relevant site-directed mutagenesis and their biochemical characterization.
authors.affiliation: State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan 250100, China. n.zhou@pentium.whiov.ac.cn.
authors.forename: Jing
authors.initials: J
authors.lastname: Su
authors.forename: Cong
authors.initials: C
authors.lastname: Zhang
authors.forename: Jun-Jie
authors.initials: JJ
authors.lastname: Zhang
authors.forename: Tiandi
authors.initials: T
authors.lastname: Wei
authors.forename: Deyu
authors.initials: D
authors.lastname: Zhu
authors.forename: Ning-Yi
authors.initials: NY
authors.lastname: Zhou
authors.forename: Li chuan
authors.initials: Lc
authors.lastname: Gu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001053:Apolipoproteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D019143:Evolution, Molecular; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006801:Humans; D008298:Maleates / Q000378:metabolism; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009596:Nitrophenols / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011549:Pseudomonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001053:Apolipoproteins; D001426:Bacterial Proteins; D005231:Fatty Acids, Unsaturated; D008298:Maleates; D009596:Nitrophenols; D009243:NAD; C011905:maleoylacetic acid; C002091:hydroxymuconic semialdehyde; D010088:Oxidoreductases; C024836:4-nitrophenol