Article Cite This: Biochemistry XXXX, XXX, XXX−XXX
pubs.acs.org/biochemistry
Crystal Structure of Aeromonas hydrophila Cytoplasmic 5′Methylthioadenosine/S‑Adenosylhomocysteine Nucleosidase Jinli Chen,†,‡,∇ Wei Liu,†,‡ Lulu Wang,†,‡,§ Fei Shang,†,‡ Yuanyuan Chen,†,‡ Jing Lan,†,‡ Peng Gao,∥ Nam-Chul Ha,⊥ Chunshan Quan,*,†,‡ Ki Hyun Nam,*,#,@ and Yongbin Xu*,†,‡ †
Department of Bioengineering, College of Life Science, Dalian Minzu University, Dalian 116600, Liaoning, China Key Laboratory of Biotechnology and Bioresources Utilization, Dalian Minzu University, Ministry of Education, Dalian 116600, China § School of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, Liaoning, China ∥ Clinical Laboratory, Dalian Sixth People’s Hospital, Dalian 116024, Liaoning, China ⊥ Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Gwanak-gu, Seoul 08826, Republic of Korea # Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea @ Institute of Life Science and Natural Resources, Korea University, Seoul 02841, Republic of Korea
Downloaded via BUFFALO STATE on July 22, 2019 at 03:03:38 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
‡
S Supporting Information *
ABSTRACT: 5′-Methylthioadenosine/S-adenosyl-L-homocysteine (MTA/SAH) nucleosidase (MTAN) is an important enzyme in a number of critical biological processes. Mammals do not express MtaN, making this enzyme an attractive antibacterial drug target. In pathogen Aeromonas hydrophila, two MtnN subfamily genes (MtaN-1 and MtaN-2) play important roles in the periplasm and cytosol, respectively. We previously reported structural and functional analyses of MtaN-1, but little is known regarding MtaN-2 due to the lack of a crystal structure. Here, we determined the crystal structure of cytosolic A. hydrophila MtaN-2 in complex with adenine (ADE), which is a cleavage product of adenosine. AhMtaN-1 and AhMtaN-2 exhibit a high degree of similarity in the α−β−α sandwich fold of the core structural motif. However, there is a structural difference in the nonconserved extended loop between β7 and α3 that is associated with the channel depth of the substratebinding pocket and dimerization. The ADE molecules in the substrate-binding pockets of AhMtaN-1 and AhMtaN-2 are stabilized with π−π stacking by Trp199 and Phe152, respectively, and the hydrophobic residues surrounding the ribose-binding sites differ. A structural comparison of AhMtaN-2 with other MtaN proteins showed that MtnN subfamily proteins exhibit a unique substrate-binding surface and dimerization interface.
T
adhesin molecules, and cytokine secretion.9,10 Therefore, breakdown of MTA and SAH is critical for the regulation of cellular processes in bacteria as well as in mammalian cells. Thus, MtaNs have been regarded as potential targets for antibacterial drug design. Aeromonas hydrophila is a motile, Gram-negative, facultatively anaerobic, rod-shaped bacterium of the family Vibrionaceae that is pathogenic for fish, frogs, and mammals.11 Unlike other bacteria, A. hydrophila has two MtnN subfamily proteins: periplasmic MtaN-1 and cytosolic MtaN-2.12,13 We previously showed significant differences between these two proteins, in that AhMtaN-1 exhibits an inherent trypsin
he enzyme 5′-methylthioadenosine/S-adenosyl-L-homocysteine (MTA/SAH) nucleosidase (MTAN) belongs to the MtnN subfamily of the purine nucleoside phosphorylase/ uridine phosphorylase (PNP/UDP) family, which plays a key role in four metabolic processes: polyamine biosynthesis,1 methylation of DNA and other macromolecules,2 methionine recycling,3 and bacterial quorum sensing.4,5 In bacterial cells, this enzyme catalyzes the irreversible cleavage of the Nglycosidic bonds of 5′-methylthioadenosine (MTA), 5′deoxyadenosine (5′-DAO), and S-adenosylhomocysteine (SAH), generated from various S-adenosylmethionine (SAM) radical reactions, to form adenine (ADE), and S-methyl-5′thioribose (MTR), 5′-deoxyribose, and S-ribosyl-L-homocysteine (SRH), respectively.6−8 In mammalian species, the absence of MtaN homologue proteins (MTA phosphorylase and SAH hydrolase) leads to the accumulation of MTA and SAH, perturbing cAMP metabolism, endothelial expression of © XXXX American Chemical Society
Received: March 1, 2019 Revised: June 17, 2019 Published: July 5, 2019 A
DOI: 10.1021/acs.biochem.9b00174 Biochemistry XXXX, XXX, XXX−XXX
Article
Biochemistry resistance that is higher than that of AhMtaN-2, whereas AhMtaN-2 exhibits a thermal stability that is higher than that of AhMtaN-1, according to the results of in vitro experiments.13 We determined the crystal structure of AhMtaN-1, which exhibited structural similarity with the nucleoside phosphorylase-1 (NP-1) family of enzymes.13 Moreover, we determined the crystal structures of AhMtaN-1 in complex with the substrates SAH, MTA, and 5′-DAO to gain insight into substrate recognition.13 Structural analysis and enzymatic assays identified that, in substrate recognition by AhMtaN-1, the nonconserved amino acid Trp199 plays a crucial role in substrate binding.13 Therefore, the function of periplasmic MtaN-1 has been well characterized, whereas the structurebased molecular function of cytoplasmic MtaN-2 remains largely uncharacterized. To better understand the molecular function of AhMtaN-2, we determined the crystal structure of cytosolic MtaN-2 from A. hydrophila in complex with ADE. We describe the dimer formation and active site pocket of AhMtaN-2. Differences in the extended loop region and active site were observed via structural comparison between AhMtaN-1 and AhMtaN-2. In addition, AhMtaN-2 was compared with other MtaN proteins to gain insights into its functional mechanisms. Our studies provide novel insight into the mechanism of action of MtnN subfamily proteins.
■
Table 1. Crystallography Data and Refinement Statistics beamline resolution range (Å) space group total/unique reflections a, b, c (Å) α, β, γ (deg) Rsym (%) completeness (%) multiplicity average I/σ(I)
SSRF BL17U1 32.45−2.0 (2.07−2.00)a P32 76730/7397 74.94, 74.94, 185.21 90.00, 90.00, 120.00 18.5 (55.0) 97.68 (93.95) 4.0 (3.1) 17.07 (3.69) Model Refinement Rfactor/Rfreeb (%) 15.30 (24.79)/19.05 (28.22) no. of protein atoms 6788 no. of water molecules 680 average B factor (Å2) 39.80 root-mean-square deviation for bonds (Å) 0.013 root-mean-square deviation for angles (deg) 1.40 Ramachandran preferred (%) 97 Ramachandran outliers (%) 0.11 Ramachandran allowed (%) 2.89 PDB entry 6K2Q a Values in parentheses refer to the highest-resolution shell. b∑hkl|Fo − Fc|/∑hkl|Fo| for all data with Fo > 2σ(Fo), excluding data used to calculate Rfree. Rfree = ∑hkl|Fo − Fc|/∑hkl|Fo| for all data with Fo > 2σ(Fo) that were excluded from the refinement.
MATERIALS AND METHODS
Overexpression and Purification of Recombinant AhMtaN-2 Protein. The full sequence encoding A. hydrophila MtaN-2 (UniProt entry A0A2R7N3M3, residues 1−230) was cloned between the EcoRI and HindIII sites of the pPROTEXHTA vector (Invitrogen). The expression and purification of native MtaN-2 have been previously described.14 To obtain protein samples of AhMtaN-2 in complex with adenosine (ADO), ADO was added to cell lysis buffer and gel filtration buffer to a final concentration of 2 mM. The purified proteins were concentrated to 10 mg mL−1 using Centriprep columns (Millipore) for crystallization and stored frozen at 193 K until use. The purity of the produced protein was confirmed by 15% sodium dodecyl sulfate−polyacrylamide gel electrophoresis and Coomassie blue staining. Crystallization, X-ray Data Collection, and Structure Determination. Crystallization and preliminary X-ray analysis of AhMtaN-2 have been published previously.14 Initial phases were obtained via molecular replacement15 using the PHENIX package16 with the VcMtaN structure [Protein Data Bank (PDB) entry 4WKB]17 as a search model. The refined models were further improved by visual inspection and several rounds of manual model building using the program COOT18 followed by several cycles of refinement with PHENIX.REFINE, implemented in PHENIX.19 The final models were evaluated with MolProbity.20 Data collection and refinement statistics are listed in Table 1. Pictures of the structural models were generated using PyMOL.21 The PISA server from EBIEMBL was used to calculate the interface areas of the oligomer.22 Multiple-sequence alignments were generated using the Clustal Omega server and visualized using ESPript 3.0.23
functions and mechanisms of A. hydrophila MtaNs, we performed a crystallographic study of cytosolic MtaN-2 from A. hydrophila. We first attempted to determine the crystal structure of apo AhMtaN-2 and successfully obtained crystals; however, the crystals exhibited poor diffraction. Instead, we collected diffraction data of AhMtaN-2 in complex with ADE by adding ADO during protein purification. The crystal of AhMtaN-2 belongs to trigonal space group P32, with unit cell parameters a = b = 74.94 Å and c = 185.21 Å, and contains four molecules in the asymmetric unit. The refined model was determined to a resolution of 2.0 Å with Rfactor and Rfree values of 0.153 and 0.191, respectively. The electron density map of AhMtaN-2 was well defined for all residues except those between Ile133 and Lys141 in the disordered region (Figure 1A). The monomeric structure of AhMtaN-2 has an α−β−α sandwich fold in which 10 β-strands form a central parallel βsheet surrounded by six α-helices and a small three-helix bundle and a subdomain composed of a twisted loop (Figure 1B). This α−β−α sandwich fold is identical to a common core structural motif that is found in all MtnN subfamily proteins.8,17,26,27 Superimposition of four molecules in the asymmetric unit shows a high degree of similarity, with a rootmean-square deviation (rmsd) of
