This is the publisher's version, also available electronically from http://scripts.iucr.org/cgi-bin/paper?S0907444999010318.Guanidinoacetate methyltransferase is the enzyme which catalyzes the last step of creatine biosynthesis. The enzyme is found ubiquitously and in abundance in the livers of all vertebrates. Recombinant rat-liver guanidinoacetate methyltransferase has been crystallized with guanidinoacetate and S-adenosylhomocysteine. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 54.8, b = 162.5, c = 56.1 Å, [beta] = 96.8 (1)° at 93 K, and typically diffract beyond 2.8 Å

Fujioka, Motoji

Gomi, Tomoharu

Hu, Yongbo

Huang, Yafei

Komoto, Junichi

Konishi, Kiyoshi

Ogawa, Hirofumi

Takata, Yoshimi

Takusagawa, Fusao

Acta Crystallographica

English

KU ScholarWorks

1928 Komoto et al.  Guanidinoacetate methyltransferase Acta Cryst. (1999). D55, 1928±1929crystallization papersActa Crystallographica Section DBiologicalCrystallographyISSN 0907-4449Crystallization and preliminary X-ray diffractionstudies of guanidinoacetate methyltransferase fromrat liverJunichi Komoto,a Yafei Huang,aYongbo Hu,a Yoshimi Takata,bKiyoshi Konishi,b HirofumiOgawa,b Tomoharu Gomi,bMotoji Fujiokab and FusaoTakusagawaa*aDepartment of Molecular Biosciences, Univer-sity of Kansas, Lawrence, KS 66045-0045, USA,and bDepartment of Biochemistry, ToyamaMedical and Pharmaceutical University, Facultyof Medicine, Sugitani, Toyama 930-01, JapanCorrespondence e-mail: xraymain@ukans.edu# 1999 International Union of CrystallographyPrinted in Denmark ± all rights reservedGuanidinoacetate methyltransferase is the enzyme which catalyzesthe last step of creatine biosynthesis. The enzyme is foundubiquitously and in abundance in the livers of all vertebrates.Recombinant rat-liver guanidinoacetate methyltransferase has beencrystallized with guanidinoacetate and S-adenosylhomocysteine. Thecrystals belong to the monoclinic space group P21, with unit-cellparameters a = 54.8, b = 162.5, c = 56.1 AÊ ,  = 96.8 (1) at 93 K, andtypically diffract beyond 2.8 AÊ .Received 27 April 1999Accepted 27 July 19991. IntroductionGuanidinoacetate methyltransferase (S-adenosyl-l-methionine±guanidinoacetate N-methyl-transferase, GAMT, E.C. 2.1.1.2), ®rst found inpig liver by Cantoni & Vignos (1954), is theenzyme which catalyzes the last step of crea-tine biosynthesis. The enzyme is foundubiquitously and in abundance in the livers ofall vertebrates.In humans, the biosynthesis of creatine isreported to represent about 75% of the totalutilization of methionine through S-adenosyl-methionine (AdoMet; Mudd & Poole, 1975);GAMT is therefore believed to be the majorenzyme involved in the metabolic conversionof AdoMet to S-adenosylhomocysteine(AdoHcy) in vertebrates. A hereditary diseasewith extrapyramidal motor disorder andextremely low concentrations of creatine in thebrain, serum and urine has recently beendescribed (StoÈ ckler et al., 1994) and shown tobe a consequence of a de®ciency of GAMT inthe liver (StoÈ ckler et al., 1996, 1997).GAMT was puri®ed to homogeneity fromthe livers of pig and rat by Im et al. (1979) andOgawa et al. (1983), respectively, and shown tobe a monomeric protein with a relatively smallmolecular size (Mr = 26 kDa). The amino-acidsequences of the rat (Ogawa et al., 1988) andhuman enzymes (Isbrandt & Figura, 1995)were deduced from the respective cDNAsequences (Ogawa et al., 1988; Isbrandt &Figura, 1995), respectively. There is a 82.5%homology in the nucleotide sequence and a86.9% homology in the deduced amino-acidsequence between the two enzymes. The rat-liver GAMT was produced recombinantly inlarge amounts in Escherichia coli (Ogawa et al.,1988) and its structural and functional featureshave been studied by chemical modi®cation,site-directed mutagenesis and limited proteo-lysis. These studies have revealed that Cys15,Cys90 and Cys219 occur spatially closetogether (Fujioka et al., 1988; Takata et al.,1991) and that the region around residues19±24 is highly exposed to the solvent and is¯exible (Takata & Fujioka, 1990; Fujioka et al.,1991). The portion of the enzymeincluding these cysteine residues andthe N-terminal region are apparentlydistant from the active site. On theother hand, ultraviolet irradiation ofthe enzyme in the presence of AdoMetresulted in covalent attachment of thecompound to Tyr136 showing characteristics ofaf®nity labeling (Takata & Fujioka, 1992).Thus, Tyr136 occurs at or near the active site. Asubsequent site-directed mutagenesis study hasindicated that Asp134 is critical for AdoMetbinding and that Tyr136 has no direct role inthe AdoMet binding (Takata et al., 1994). Here,we describe the puri®cation, crystallization andpreliminary crystallographic characterizationof GAMT from rat liver.2. Materials and methods2.1. Puri®cationRecombinant guanidinoacetate methyl-transferase was produced in E. coli JM109transformed with plasmid pUCGAT9-1 whichcontained the coding region of rat GAMTcDNA linked to the lac promoter (Ogawa etal., 1988). The cells were grown in 2YTmedium containing 35 mg mlÿ1 ampicillin at309 K. When the cell turbidity measured at600 nm reached an absorbance of about 0.2,isopropyl-1-thio--d-galactopyranoside wasadded to a concentration of 1 mM and culturewas continued for an additional 16 h.Acta Cryst. (1999). D55, 1928±1929 Komoto et al.  Guanidinoacetate methyltransferase 1929crystallization papersCells harvested by centrifugation weresuspended in 25 ml of 50 mM Tris±HCl pH7.5 containing 2 mM EDTA and disruptedby treatment with egg-white lysozyme(1 mg mlÿ1) for 30 min in ice. Followingfreeze-thawing and brief sonication, the celldebris was removed by centrifugation. Thesupernant was then treated with ammoniumsulfate and the precipitate obtained between40 and 55% saturation was dissolved in 1 mlof a buffer solution containing 10 mM Tris±HCl pH 7.5, 1 mM EDTA and 1 mMdithiothreitol. The solution was directlyloaded onto a column of Sephacryl S200(32  980 mm) equilibrated and eluted withthe same buffer. The active fractions werecombined and applied to a column ofDEAE-cellulose (DE-52, 32  250 mm)which had been equilibrated with 10 mMTris±HCl pH 7.5, 1 mM EDTA. The enzymewas eluted with a linear gradient between60 ml each of solution A (10 mM Tris±HClpH 7.5, 1 mM EDTA, 1 mM dithiothreitol)and solution B (70 mM potassium phosphatepH 7.2, 1 mM EDTA, 1 mM dithiothreitol).The enzyme was concentrated by ultra-®ltration. The homogeneity of the enzymewas checked by SDS±PAGE. The recombi-nant enzyme lacks the N-terminal acyl grouppresent in the liver enzyme, but shows thesame kinetic and physical properties (Ogawaet al., 1988).2.2. CrystallizationCrystallization trials were performed atthree different temperatures [incubator(299 K), room temperature (295 K), andcold room temperature (277 K)] by thehanging-drop vapor-diffusion method.Conditions for crystallization were searchedinitially by the PEG screen method. Proteinsolutions (20 mg mlÿ1) with and withoutadded 5 mM AdoHcy were utilized in thescreening experiment. Having obtainedneedle micro-crystals of the enzyme fromthe solution containing 10% polyethyleneglycol molecular weight 4000 (PEG 4000),100 mM MES buffer (pH 6.5), the optimalconditions for growing crystals for X-raydiffraction study were searched by varyingthe pH, buffer concentration, precipitantand temperature (277±299 K). Crystalssuitable for X-ray diffraction studies (0.70 0.15  0.15 mm) were grown in a solutioncontaining 50 mM MES buffer (pH 6.5),1 mM dithiothreitol, 5 mM AdoHcy, 20 mMguanidinoacetate, 8%(w/v) PEG 4000 with aprotein concentration of 10 mg mlÿ1 in a277 K cold room (Fig. 1).2.3. X-ray diffraction experimentThe tetragonal prism-like crystals ofGAMT diffracted well to 2.8 AÊ , but werevery sensitive to radiation; it was onlypossible to collect a few frames from eachcrystal. Conditions for cryocooling thecrystals were therefore sought in an attemptto circumvent this dif®culty. A crystal (0.1 0.1  0.5 mm) in a hanging drop wasscooped up in a nylon loop and dipped into acryoprotectant solution containing 50 mMMES buffer (pH 6.5), 1 mM dithiothreitol,5 mM AdoHcy and 20%(v/v) glycerol for30 s before being ¯ash-frozen in coldnitrogen gas (93 K) on a Rigaku R-AXIS IIcimaging-plate X-ray diffractometer with arotating-anode X-ray generator as an X-raysource (Cu K radiation generated at 50 kVand 100 mA). The diffraction data weremeasured to 2.8 AÊ resolution (Fig. 2). Thedata were processed with the programDENZO (Otwinowski & Minor, 1997). Thespace group and unit-cell parameters are P21and a = 54.8, b = 162.5, c = 56.1 AÊ ,  = 96.8,respectively. From 72400 re¯ectionsrecorded on the detector, 23515 indepen-dent re¯ections (96.8% complete) wereobtained with Rsym ( =P jI ÿ hIij=P jIj) =0.054. The data statistics are given in Table 1.The calculated unit-cell volume of496961 AÊ 3 suggests that there are fourmonomeric GAMT molecules in the asym-metric unit, with a Vm of 2.39 AÊ3 Daÿ1,corresponding to a solvent content of 48%(Matthews, 1968). We have started screeningpossible heavy-atom derivatives. The struc-tural determination of GAMT will bereported in the future.This research has been supported by theNIH grant GM37233.ReferencesCantoni, G. L. & Vignos, P. J. Jr (1954). J. Biol.Chem. 209, 647±659.Fujioka, M., Konishi, K. & Takata, Y. (1988).Biochemistry, 27, 7658±7664.Fujioka, M., Takata, Y. & Gomi, T. (1991). Arch.Biochem. Biophys. 285, 181±186.Im, Y. S., Chiang, P. K. & Cantoni, G. L. (1979). J.Biol. Chem. 254, 11047±11050.Isbrandt, D. & von Figura, K. (1995). Biochim.Biophys. Acta, 1264, 265±267.Matthews, B. W. (1968). J. Mol. Biol. 33, 491±497.Mudd, H. S. & Poole, J. R. (1975). Metabolism, 24,721±735.Ogawa, H., Date, T., Gomi, T., Konishi, K., Pitot,H. C., Cantoni, G. L., & Fujioka, M. (1988).Proc. Natl Acad. Sci. USA, 85, 694±698.Ogawa, H., Ishiguro, Y. & Fujioka, M. (1983).Arch. Biochem. Biophys. 226, 265±275.Otwinowski, Z. & Minor, W. (1997). MethodsEnzymol. 276, 307±326.StoÈ ckler, S., Holzbach, U., Hanefeld, F.,Marquardt, I., Helms, G., Requart, M., HaÈnicke,W. & Frahm, J. (1994). Pediatr. Res. 36, 409±413.StoÈ ckler, S., Isbrandt, D., Hanefeld, F., Schmidt, B.& von Figura, K. (1996). Am. J. Hum. Genet. 58,914±922.StoÈ ckler, S., Marescau, B., De Deyn, P. P., Trijbels,J. M. & Hanefeld, F. (1997). Metabolism, 46,1189±1193.Takata, Y., Date, T. & Fujioka, M. (1991).Biochem. J. 277, 399±4069.Takata, Y. & Fujioka, M. (1990). Int. J. Biochem.22, 1333±1339.Takata, Y. & Fujioka, M. (1992). Biochemistry, 31,4369±4374.Takata, Y., Konishi, K., Gomi, T. & Fujioka, M.(1994). J. Biol. Chem. 269, 5537±5542.Figure 1Crystals of guanidinoacetate methyltransferase withguanidinoacetate and S-adenosylhomocysteine.Figure 2Diffraction pattern from a 1 oscillation image of themonoclinic crystal with 15 min exposure. The crystal-to-detector distance was 160 mm; the resolution atthe edge of the image is 2.8 AÊ .Table 1Data statistics.ResolutionrangeNumber ofre¯ectionsComplete-ness (%) I/ Rsym99.00±6.01 2437 98.0 18.1 0.0536.01±4.77 2419 99.5 17.9 0.0494.77±4.17 2386 98.6 17.8 0.0494.17±3.79 2387 98.0 17.3 0.0523.79±3.52 2385 98.8 16.8 0.0563.52±3.31 2394 98.8 15.7 0.0643.31±3.14 3413 99.4 14.5 0.0733.14±3.01 2404 99.3 13.0 0.0873.01±2.89 2393 99.3 11.7 0.1102.89±2.79 1897 78.1 7.5 0.128All re¯ections 23515 96.8 15.0 0.059

Crystallization and preliminary X-ray diffraction studies of guanidinoacetate methyltransferase from rat liver

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Crystallization and preliminary X-ray diffraction studies of guanidinoacetate methyltransferase from rat liver

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