Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

NW12/2005G048

Crystal structure of 5’-methylthioadenosine nucleosidase from Arabidopsis
thaliana
Eun Young PARK, Byung-Gil LEE, Byung-Cheon Jeong, and Hyun Kyu SONG*
School of Life Sciences and Biotechnology, Korea University, Anam-Dong
Seongbuk-Gu, Seoul 136-701, South Korea
Introduction
Methylthioadenosine(MTA;5’-deoxy-5’methylthioadenosine) is an important metabolite in allliving organisms. The MTA cycle has been studied
extensively in plant because of the synthesis of ethylene
and polyamines, where the maintenance of their levels is
important in plant physiology [1]. Many genes involvedin the MTA cycle in plant have been isolated andcharacterized. One of the key enzymes in this cycle is 5’methylthioadenoside nucleosidase (MTAN).
MTAN (EC 3.2.2.16) is an enzyme that
irreversibly cleaves the ribosidic bond of MTA toproduce 5’-methylthioribose (MTR) and adenine.
Extensive biochemical and structural studies on E. coli
MTAN have provided a wealth of information regarding
the catalytic mechanism, specific interactions with MTA,
and conformational changes for the enzymatic reaction[2]. No structural information is available, however, on aplant MTAN, a similar enzyme with different specificity.
To gain insight into the structural basis of the differencesin substrate specificity, we have determined the highresolution structure of MTAN from Arabidopsis thaliana(AtMTAN).
Materials and methods
The purified MTAN was concentrated to 10
mg/mL in 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1mM DTT. Crystallization was performed by the hangingdrop vapor diffusion method at 22°C. A monoclinic
crystal form of MTAN was obtained with a reservoir
solution consisting of 100 mM Tris-HCl, pH = 8.5, 30 %(w/v) polyethylene glycol 3000, and 200 mM sodium
acetate. Crystals grew to a maximum size of 0.5 × 0.3 ×0.2 mm3 within 2 days. The native diffraction data werecollected on a charge-coupled device detector at theNW12 beamline of the Photon Factory, Tsukuba, Japan.
All diffraction data were processed and scaled using theHKL2000 software package.
Eight selenium and two mercury sites in theasymmetric unit were located with SOLVE. The phases
were improved with RESOLVE. The electron density wasof sufficient quality to build a nearly complete model of
MTAN with the guidance of EcMTAN coordinates (PDBID: 1Z5P). The protein model was refined with CNS
including the bulk solvent correction. A two-fold noncrystallographic symmetry was maintained with tightrestraint during the early stages of refinement and wasrelaxed in the final rounds. Solvent molecules were addedusing model-phased difference Fourier maps. An
unambiguous electron density of adenine was visible,
even in the initial MAD-phased map, although the MTAN
crystals were obtained in the absence of adenine. The
assessment of model geometry and the assignment of
secondary structural elements were obtained by using theprogram PROCHECK.
Results
The crystal structure of AtMTAN has beenrefined at 1.5 Å resolution. Each monomer of theAtMTAN dimer consisted of seven α-helices, ten βstrands, and two 310-helices (Figure 1). The first 21
residues (Met1-Glu21) of AtMTAN were not observed in
the electron density and, therefore, are likely unstructured.
The location of bound adenine is in the deep pocket
formed by a monomer and the entrance is partiallycovered by the adjacent subunit (Figure 1). This flap isimportant for forming a wide dimeric interface. Structuralsimilarities of AtMTAN with EcMTAN and human
MTAP (HsMTAP) are anticipated on the basis ofsequence homology, the utilization of the same substrate(MTA) or a combination of the two.

Figure 1. Overall structure of the AtMTAN. (A) Ribbon
diagram showing the dimeric AtMTAN structure and (B)a view along the non-crystallographic two-fold molecularsymmetry axis. Each monomer in AtMTAN is coloredyellow and magenta, respectively. The bound adenine
molecules are shown in the stick model. The N- and Ctermini of AtMTAN are labeled.
References[1] J.H. Miyazaki et al., Physiol. Plant 69, 366-70 (1987).
[2] J.E. Lee et al., J. Mol. Biol. 352, 559-74 (2005).
* hksong@korea.ac.kr

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