USOO59 19673A United States Patent (19) 11 Patent Number: 5,919,673 Wong et al. (45) Date of Patent: Jul. 6, 1999 54) ONE-POTENZYMATIC SULFATION Sekura, Methods in Enzymology, 77:413 (1981. PROCESS USING 3'-PHOSPHOADENOSINE Horwitz et al., Biochem. Biophy. Acta., 480:376 (1977). 5'-PHOSPHOSULEATE AND RECYCLED Satishchandran et al., J. Biol. Chem., 264:15012 (1989). PHOSPHORYLATED ADENOSINE Fernando et al., Biosci. Biotech. Biochem, 57:1974 (1993). INTERMEDIATES Mukai, Agric. Biol. Chem, 53:883 (1989). Leyh et al., J. Biol. Chem., 263:2409 (1988). 75 Inventors: Chi-Huey Wong, Rancho Sante Fe; Renosto et al., J. Biol. Chem., 259:2113 (1989). Chun-Hung Lin, San Diego; Marcus et al., Anal. Biochem., 107:296 (1980). GWO-Jenn Shen, Carlsbad, all of Calif. Suzuki et al., Biochim. Biophys. Acta, 50:169 (1961). Robinson, Biochem. J., 113:543 (1969). 73 Assignee: The Scripps Research Institute, Kimata et al., Mol. Cell. Biochem. 1:211(1973). LaJolla, Calif. Silbert, J. Biol. Chem., 239:1310 (1964). Kim et al., J. Biol. Chem., 252:8292 (1977). 21 Appl. No.: 08/408,774 Habuchi et al., Biochim. Biophys. Acta, 208:616 (1980). Habuchi et al., Biochim. Biophys. Acta, 414:717 (1982). 22 Filed: Mar. 22, 1995 Habuchi et al., J. Biol. Chem., 246:7357 (1971). 51) Int. Cl. ............................. C12P 11/00; C12P33/00; Nakanishi et al., J. Biol. Chem., 256:5443 (1981). C12P 19/04; C12P 19/32 Sugumaran et al., J. Biol. Chem., 261: 12659 (1986). 52 U.S. Cl. ............................ 435/130; 435/101; 435/52; Fisher et al., Nature, 357:655 (1992). 435/92 LeRouge et al., Nature, 344:781 (1990). Faucher et al., Mol. Plant-Microbe Interactions, 2:291 58 Field of Search ..................................... 435/130, 101, (1989). 435/52, 92 Faucher et al., J. Bacteriol., 170:5489 (1988). 56) References Cited Atkinson et al., Proc. Natl. Acad. Sci., USA, 91:8418 (1994). Schwedock et al., Mol. Plant-Microbe Interactions, 2:181 U.S. PATENT DOCUMENTS (1989). Cervantes et al., Mol. Microbiol, 3, 745 (1989. 5,376,535 12/1994 Onda et al. ............................... 435/92 Roche et al., Cell, 67: 1131 (1991). OTHER PUBLICATIONS Seubert et al., Arch. Biochem. Biophy.s., 115:679 (1983). Brunngraber, J. Biol. Chem. 233(2):472–477 (1958). Renosto et al., J. Biol. Chem., 264:9433 (1989). Kopp et al., J. Biol. Chem. 247(11):3564–3570 (1972). Burnell et al., Anal. Biochem, 68:281 (1975). Varki, Glycobiology, 3:97 (1993). Ogura et al., Molecular Pharmacol., 27:848 (1990). Yuen et al., J. Biol. Chem., 269:1595 (1994). Primary Examiner John W. Rollins Hemmerich et al., Biochemistry, 33:4830 (1994). ASSistant Examiner-Francisco C. Prats Varki, Proc. Natl. Acad. Sci., 91:7390 (1994). Attorney, Agent, or Firm Welsh & Katz, Ltd. van Boeckel et al., Angew. Chem. Int. Ed. Eng., 32:1671 57 ABSTRACT (1993). Bernstein et al., J. Biol. Chem., 199:745 (1952). A proceSS for the enzymatic Synthase of Sulfate esterS is Robbins et al., J. Biol. Chem., 233:686 (1958). disclosed in which intermediate phosphorylated adenosine EC 2.8.2.1-28 listed in Enzyme Nomenclature 1992, E. C. compounds are recycled to minimize enzyme inhibition. Webb, ed., Academic Press, San Diego, CA 1992, pp. Exemplary enzymes include ATP Sulfurylase, APS kinase 299-303. and a Sulfotransferase. Baddiley et al., J. Am. Chem. Soc., 1067 (1957). Cherniak et al., J. Biol. Chem., 239:2986 (1964). 6 Claims, 4 Drawing Sheets U.S. Patent Jul. 6, 1999 Sheet 1 of 4 5,919,673 E. coli DNA CYS DN5 5'ATATTGAGCTCGATCAAA SacI TACGACTTACTCACCTG PCR N-terminal amplification CYS DN3 5'GCGCAAGCTTTTATTATT Hind III Stop TATCCCCCAGCAAATC "' Hind III C-terminal Cys D and CysN genes Digest with Sac I pTrcHis A vector-- and Hind III Sac I Ligate Hind III ATG (his), EK ATP sulfurylase genes Fig. 1 pTrcCysDN10 1. LacI Amp Transform into E. coli Add IPTG Express recombinant protein U.S. Patent Jul. 6, 1999 Sheet 2 of 4 5,919,673 CYS C5 5'ATATTGAGCTCGCGCTGC E. coli DNA SacI ATGACGAAAAC N-terminal CYS C3 -Go- Air P 5'GCGCAAGCTTTTATTAGG amplification Hind III Stop ATCTGATAATATCGTT Sac I Hind III C-terminal Cys C gene Digest with Sac I pTrcHis A vector-- and Hind III Sa c I '8" HindIII ATG (his). EKAPS kinase genes Transform into E. coli NagrgExpress recombinant protein U.S. Patent Jul. 6, 1999 Sheet 3 of 4 5,919,673 Rat liver mRNA STAA5 5 ATATTGAATTC CCA EcoR GACTATACTTGGTTT N-terminal cDNA STAA3 5 ACAGGCTCGAGCTT Xho I ATTCAGACATAGAACC-terminal amplificationE. EcoRI Xho I HSSTase gene pFlag-1 vector digested with -e- EcoRI and Xho I Ligate pflaghSST-12 Transform into E. coli Add IPTG Express recombinant protein U.S. Patent Jul. 6, 1999 Sheet 4 of 4 5,919,673 NOdHST5' 5' ATATTGAATTCATTTT Rhizobium meliloti DNA ECOR I. CATGACCCATTCCA Start -- PCR amplification NOdHST3' 5' GGCGCGGATCCTTAGT ECOR I BamHI CGTTAGdAABamH Icre Stop nodH gene (NodST) Digested with EcoRI pKEN2 vector -- and BamH I Ligation - Piac IgAATTCATTTTCATGf".NodHST (750 TAAGGATCCkb) ECOR I Start Stop BamH I NodSTpKEN10 pUC ori Transform into E. coli Add IPTG Amp' Express recombinant protein 5,919,673 1 2 ONE-POTENZYMATIC SULFATION In the course of Sulfation, inorganic Sulfate is activated PROCESS USING 3'-PHOSPHOADENOSNE first, followed by a transfer of the sulfate group to the final 5'-PHOSPHOSULEATE AND RECYCLED PHOSPHORYLATED ADENOSINE acceptor Bernstein et al., J. Biol. Chem., 199:745 (1952)). INTERMEDIATES 5 The two key enzymes involved in this activation process are ATP sulfurylase (EC 2.7.7.4) and adenosine-5'- DESCRIPTION phosphosulfate (APS) kinase (EC 2.7.1.25) Robbins et al., 1. Technical Field J. Biol. Chem., 233:686 (1958); their reactions are shown in The present invention relates to the enzymatic synthesis Scheme 1, below, in which “A” is adenosine, “ROH' is the of Sulfate esters, and more particularly to a single vessel acceptor, and "PPi' is pyrophosphate, following usually enzyme-catalyzed Sulfation of an acceptor molecule by used abbreviations. Scheme 1 O-P-O-P-O-P-OI A. O-S-O-P-OM A. O ATP Sulfurylase O O O O + SO’ s =s O O - PP HO OH HO OH ATP APS O-S-O-P-O| M ADP- O O O O APS Kinase -- OH O PAPS 45 3'-phosphoadenosine-5'-phosphoSulfate in which intermedi 3'-Phosphoadenylsulfate, also known as ate phosphorylated adenosine compounds are recycled. 3'-phosphoadenosine-5'-phosphosulfate (PAPS), is the prod uct generated in these two enzymatic reactions and is a 2. Background Art Substrate and cofactor for the enzymatic Sulfation of oli 50 gosaccharides and Steroids using Sulfotransferases. Of the Sulfated biomolecules play important roles in many bio twenty-eight Sulfotransferase enzymes EC 2.8.2.1-28 logical processes Varki, Glycobiology, 3:97 (1993)). For listed in Enzyme Nomenclature 1992, E. C. Webb, ed., example, the Sulfated Lea tetra- and pentasaccharides Yuen Academic Press, San Diego, Calif. 1992, pages 299-303, all et al., J. Biol. Chem., 269:1595 (1994) are potent E-selectin but one enzyme utilize PAPS as the Sulfate donor. inhibitors and Sialyl Lewis X with a Sulfate group at the 55 Several groups have reported the synthesis of PAPS 6-position of galactose is a ligand for L-selectin Baddiley et al., J. Am. Chem. Soc., 1731 (1959); Cherniak et al., J. Biol. Chem., 239:1986 (1964); Sekura, Methods in Hemmerich et al., Biochemistry, 33:4830 (1994). These Enzymology, 77:413 (1981); Horwitz et al., Biochem. Sulfated Sugars play important roles in cell adhesion in Biophy, Acta., 480:376 (1977); Satishchandran et al., J. Biol. response to inflammatory reactions Varki, Proc. Natl. Acad. 60 Chem., 264:15012 (1989); Fernando et al., Biosci. Biotech. Sci., 91:7390 (1994)). Many glycosaminoglycans are also Biochem., 57:1974 (1993); Mukai, Acric. Biol. Chem. Sulfated and are involved in numerous cellular functions 53:883 (1989)). The procedures involve many steps and gave very low yields Baddiley et al., J. Am. Chem. Soc., van Boeckel et al., Angew. Chem. Int. Ed. Eng., 32:1671 1731 (1959); Cherniak et al., J. Biol. Chem., 239:1986 (1993)). In addition, the sulfation of hydroxysteroids pro 65 (1964); Sekura, Methods in Enzymology, 77:413 (1981); vides hydrophilic forms for excretion Ogura et al., Bio Horwitz et al., Biochem. Biophy, Acta., 480:376 (1977). The chem. Biophys. Res. Commun., 165: 169 (1989)). enzymatic preparation of PAPS on nmol to limol Scales 5,919,673 3 4 using isolated enzymes was described previously The aqueous reaction medium is maintained at a pH value Satishchandran et al., J. Biol. Chem., 264:15012 (1989); of about 5 to about 10 at a temperature of about Zero degrees Fernando et al., Biosci. Biotech. Biochem., 57:1974 (1993); C to about 40° C. for a time period sufficient for the acceptor Mukai, Agric. Biol. Chem. 53:883 (1989); however, it was to be sulfated. The sulfated acceptor is thereafter preferably not clear if the proceSS was feasible for larger Scale Synthe recovered. SS. A process of Scheme 3, hereinafter, for using APS to Recently, the genes coding for ATP Sulfurylase and APS prepare PAPS with recycling of ATP and ADP thus com kinase in E. coli have been identified Leyh et al., J. Biol. prises the Steps of Chem., 263:2409 (1988). These genes are located in the (a) admixing the following ingredients in an aqueous cluster of cys CDHIJ of E. coli. ATP Sulfurylase contains medium containing magnesium and potassium ions two different Subunits corresponding to two genes, cySD within a single vessel to form an aqueous reaction and cys N. APS kinase contains one Subunit corresponding medium to the cySC gene. (i) ATP Sulfurylase; ATP is a Substrate for ATP Sulfurase and is a cofactor/ (ii) APS kinase; substrate for APS kinase.