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Structural Insights Into Decreased Enzymatic Activity Induced by an Insert Sequence in Mannonate Dehydratase from Gram Negative Bacterium

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Structural Insights Into Decreased Enzymatic Activity Induced by an Insert Sequence in Mannonate Dehydratase from Gram Negative Bacterium Journal of Structural Biology 180 (2012) 327–334 Contents lists available at SciVerse ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi Structural insights into decreased enzymatic activity induced by an insert sequence in mannonate dehydratase from Gram negative bacterium Xiaoting Qiu, Yuyong Tao, Yuwei Zhu, Ye Yuan, Yujie Zhang, Hejun Liu, Yongxiang Gao, ⇑ ⇑ Maikun Teng , Liwen Niu 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 230026, PR China article info abstract Article history: Mannonate dehydratase (ManD; EC4.2.1.8) catalyzes the dehydration of D-mannonate to 2-keto-3-deoxyg- Received 30 April 2012 luconate. It is the third enzyme in the pathway for dissimilation of D-glucuronate to 2-keto-3-deoxygluc- Received in revised form 21 June 2012 onate involving in the Entner–Doudoroff pathway in certain bacterial and archaeal species. ManD from Accepted 26 June 2012 Gram negative bacteria has an insert sequence as compared to those from Gram positives revealed by Available online 14 July 2012 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 Keywords: reveal that this insert sequence forms two a helices locating above the active site. The two insert a helices Mannonate dehydratase introduce a loop that forms a cap covering the substrate binding pocket, which restricts the tunnels of sub- Gram negative bacteria Insert sequence strate entering and product releasing from the active site. Site-directed mutations and enzymatic activity Substrate entering tunnel assays confirm that the catalytic rate is decreased by this loop. These features are conserved among Gram Glucuronate metabolism negative bacteria. Thus, the insert sequence of ManD from Gram negative bacteria acts as a common indu- Entner–Doudoroff pathway cer 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. Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved. 1. Introduction species (Mekjian et al., 1999; Rakus et al., 2007; Robert-Baudouy et al., 1982; Shulami et al., 1999). ManD is the third enzyme in D-glucuronate is a six-carbon acidic sugar that is abundantly the pathway for dissimilation of D-glucuronate to 2-keto-3-deoxyg- distributed in carbohydrate chain of proteoglycan in many animal luconate (2-KDG) involving in the Entner–Doudoroff pathway and plant tissues (Kuehl and Murphy, 2003; Murarka et al., 2010), (Murarka et al., 2010). In this step, ManD catalyzes the removal of where it serves as the sole carbon source of several endogenous one water molecule from C2-hydroxyl and C3-hydroxyl of bacterial species (Lawford and Rousseau, 1997). All macromolecu- D-mannonate to yield 2-KDG (Rakus et al., 2007; Zhang et al., lar components required by these bacteria are synthesized from 2009), which is then cleaved to pyruvic acid and glyceraldehyde D-glucuronate. Therefore, glucuronate metabolism is critical for 3-phosphate (Murarka et al., 2010). These compounds conse- the growth of these bacteria. Catabolism of D-glucuronate occurs quently participate in the tricarboxylic acid cycle and gluconeogen- via the Entner–Doudoroff pathway. Expressions of enzymes partic- esis to yield energy and precursors for polysaccharide synthesis, ipating in this pathway are induced by glucuronate and repressed respectively (Nelson and Cox, 2000). When living in its natural hab- by glucose (Mekjian et al., 1999), which suggests that the Entner– itat (the large intestine),Escherichia coli, as a model species for Gram Doudoroff pathway uses D-glucuronate as the substrate only if bac- negative bacteria, can utilize glucuronate as the sole carbon source teria live in an environment that contains glucuronate as the sole for growth. The Entner–Doudoroff pathway using D-glucuronate as carbohydrate component. the substrate is therefore critical for the colonization of E. coli in the The gene uxuA encodes mannonate dehydratase (ManD; large intestine (Peekhaus and Conway, 1998). EC4.2.1.8), which has been found in certain bacterial and archaeal Bacterial ManDs are structurally assigned to the xylose isomer- ase-like superfamily, member of which contains a modified (ba)8 Abbreviations: ManD, mannonate dehydratase; 2KDG, 2-keto-3-deoxygluconate. ⇑ Corresponding authors. Address: 96 Jinzhai Rd., School of Life Sciences, TIM barrel (Zhang et al., 2009). Sequence alignment demonstrates University of Science and Technology of China, Hefei, Anhui, PR China. that ManD from Gram negative bacteria has an insert sequence as E-mail addresses: [email protected] (M. Teng), [email protected] (L. Niu). compared to Gram positive ones. The conservation of this insert 1047-8477/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jsb.2012.06.013 328 X. Qiu et al. / Journal of Structural Biology 180 (2012) 327–334 sequence in ManD from Gram negative bacteria suggests that it has polyethylene glycol 3350 at 287 K. Before data collection, the crys- a functional importance. Structures of ManDs from two Gram po- tal was quick-soaked in a cryo-protectant solution containing 25% sitive bacteria have been solved (PDB codes 3FVM and 1TZ9), but (v/v) glycerol, 0.1 M MgCl2Á6H2O, 0.1 M Tris–HCl, pH 8.5, 25% (w/v) there is no structural information on ManD from Gram negative polyethylene glycol 3350 and flash-cooled in liquid nitrogen at bacteria reported. To elucidate the impact of this insert sequence 100 K. X-ray diffraction data were collected on beamline 17U1 at of ManD from Gram negative bacteria on the catalytic activity, the Shanghai Synchrotron Radiation Facility (SSRF) using a MX- crystal structures of ManD from E. coli strain K12 (EcManD) and 225 CCD detector (MAR Research). The crystal-to-detector distance its complex with D-mannonate were determined. was kept at 270 mm and the crystal was rotated through a total of The structure reveals that two additional helices are formed by 120° with 0.2° rotation per frame over 1.2 s. The crystal diffracted the insert sequence of EcManD as compared to those from Gram to a resolution of 2.30 Å. The data were indexed, integrated and positives. As a consequence, the loop following the insert helices scaled with HKL2000 package (Otwinowski and Minor, 1997). adopts a different trajectory, which covers the enzymatic active Space group was determined as P21212 via analysis of the system- site. By mutagenesis, we confirmed that the loop restricts the tun- atic absences in the diffraction data. For collection of enzyme–sub- nels of substrate entering and product releasing from the active strate complex data, a crystal of EcManD was soaked in a solution site to achieve a low catalytic rate, which may shed light into containing 0.2 M D-mannonate, 0.1 M Tris–HCl, pH 8.5, 25% (w/v) understanding of the glucuronate metabolism related biological polyethylene glycol 3350 at 287 K for 20 sec, followed by quick- process in Gram negative bacteria. soaking in the cryo-protectant solution described above. X-ray dif- fraction data were collected on beamline 17U1 at the SSRF using an ADSC Quantum 315r detector (Area Detector System Corporation). 2. Materials and methods The crystal-to-detector distance was kept at 300 mm and the crys- tal was rotated through a total of 130° with 0.5° rotation per frame 2.1. Cloning, expression and purification over 1.0 s. The crystal diffracted to a resolution of 2.35 Å. The data were indexed, integrated with iMOSFLM (Leslie, 2006) and scaled The open reading frame of full length EcManD (GenBank acces- with Scala (Evans, 2006) from the CCP4 suite (Collaborative sion number: BAA02590.1), which was obtained via PCR from the Computational Project 4, 1994). The final statistics of data collec- E. coli strain K12 genome was cloned into pET28a (Novagen) ex- tion and processing are tabulated in Table 1. cised using NdeI and XhoI to create recombinant EcManD with an N-terminal hexahistidine tag (MGSSHHHHHHLVPRGSH). A sin- gle colony of BL21 (DE3) bacteria harboring the expression vector 2.3. Structure determination and refinement was cultured in 8 mL Luria–Bertani broth overnight, and then used to inoculate 0.8 L media containing 50 lg/mL kanamycin. Cells The highest probability of four monomers per asymmetric unit 3 À1 were grown at 310 K for 2.5 h until OD600nm reached 0.5, and then suggested a Matthews coefficient of 2.23 Å Da (Matthews, protein expression was induced for 20 h with 0.25 mM isopropyl- b-D-thiogalactoside (IPTG) at 289 K. The bacteria were harvested Table 1 and resuspended in 40 mL binding buffer (20 mM Tris–HCl, pH Data collection and refinement statistics. 8.0, 500 mM NaCl). After disrupting the cells by sonication, bacteria were centrifuged at 15200g for 0.5 h. The supernatant recovered EcManD EcManD–D- mannonate clean lysate was loaded onto Ni-NTA agarose resin (GE Healthcare), which was pre-equilibrated with binding buffer. The tagged pro- Data collection Wavelength (Å)a 0.9792 0.9792 tein was eluted with 30 mL binding buffer including 500 mM imid- Space group P21212 P21212 azole, which was then concentrated for further purification using a Unit-cell parameters (Å, °) a = 158.96, a = 159.47, Superdex 75 gel filtration chromatography column (GE Health- b = 238.46, c = 54.35 b = 238.58, c = 54.47 care), eluted with binding buffer.
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