Structural insights into decreased enzymatic activity induced by an insert sequence in mannonate dehydratase from Gram negative bacterium.
Mannonate dehydratase (ManD; EC4.2.1.8) catalyzes the dehydration of D-mannonate to 2-keto-3-deoxygluconate. It is the third enzyme in the pathway for dissimilation of D-glucuronate to 2-keto-3-deoxygluconate involving in the Entner-Doudoroff pathway in certain bacterial and archaeal species. ManD from Gram negative bacteria has an insert sequence as compared to those from Gram positives revealed by sequence analysis. To evaluate the impact of this insert sequence on the catalytic efficiency, we solved the crystal structures of ManD from Escherichia coli strain K12 and its complex with D-mannonate, which reveal that this insert sequence forms two α helices locating above the active site. The two insert α helices introduce a loop that forms a cap covering the substrate binding pocket, which restricts the tunnels of substrate entering and product releasing from the active site. Site-directed mutations and enzymatic activity assays confirm that the catalytic rate is decreased by this loop. These features are conserved among Gram negative bacteria. Thus, the insert sequence of ManD from Gram negative bacteria acts as a common inducer to decrease the catalytic rate and consequently the glucuronate metabolic rate as compared to those from Gram positives. Moreover, residues essential for substrate to enter the active site were characterized via structural analysis and enzymatic activity assays.
Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Key Laboratory of Structural Biology, Chinese Academy of Sciences, Hefei, Anhui, PR China.
Xiaoting
X
Qiu
Yuyong
Y
Tao
Yuwei
Y
Zhu
Ye
Y
Yuan
Yujie
Y
Zhang
Hejun
H
Liu
Yongxiang
Y
Gao
Maikun
M
Teng
Liwen
L
Niu
D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D005942:Gluconates / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000201:enzymology*; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013400:Sugar Acids / Q000378:metabolism; D014961:X-Ray Diffraction
D005942:Gluconates; D013400:Sugar Acids; C002957:2-keto-3-deoxygluconate; C014078:mannonate; D006836:Hydro-Lyases; C021545:mannonate dehydratase