ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 270, No. 1, April, pp. 363-373,1989 Biosynthesis of 9-P-o-Arabinofuranosyladenine: Hydrogen Exchange at C-2’ and Oxygen Exchange at C-3’ of Adenosine’ ROBERT J. SUHADOLNIK,*” SOMCHAI PORNBANLUALAP,* JOSEPH M. WU,* DAVID C. BAKER,t AND ANNA K. HEBBLERt *Department of Biochemistry, Temple University Scrod of Medicine, Philadelphia, Pennsylvania 191.40; and fDepwtment of Chxmist~, University of Alabama, Tuscaloosa, Alabama W.87 Received August 26,1988, and in revised form December 9,1988 The data presented here describe new findings related to the bioconversion of adeno- sine to 9-@-D-arabinofuranosyladenine (ara-A) by Streptmyces antibioticus by in vivo investigations and with a partially purified enzyme. First, in double label in viva experi- ments with [2’-‘“O]- and [U-14C]adenosine, the 180:14C ratio of the ara-A isolated does not change appreciably, indicating a stereospecific inversion of the C-2’ hydroxyl of adeno- sine to ara-A with retention of the “0 at C-2’. In experiments with [3’-‘*O]- and [U-‘4C]- adenosine, [U-14C]ara-A was isolated; however, the “0 at C-3’ is below detection. The adenosine isolated from the RNA from both double label experiments has essentially the same ratio of 1a0:‘4C. Second, an enzyme has been isolated and partially purified from extracts of S. antibioticus that catalyzes the conversion of adenosine, but not AMP, ADP, ATP, inosine, guanosine, or D-ribose, to ara-A. In a single label enzyme-catalyzed experi- ment with [U-14C]adenosine, there was a 9.9% conversion to [U-14C]ara-A; with [2’-3H]- adenosine, there was a 8.9% release of the C-2’ tritium from [2’-3H]adenosine which was recovered as 3H20. Third, the release of 3H as 3Hz0 from [2’-3H]adenosine was confirmed by incubations of the enzyme with 3Hz0 and adenosine. Ninety percent of the tritium incorporated into the D-arabinose of the isolated ara-A was in C-2 and 8% was in C-3. The enzyme-catalyzed conversion of adenosine to ara-A occurs without added cofactors, displays saturation kinetics, a pH optimum of 6.8, a Km of 8 X 10e4 M, and an inhibition by heavy metal cations. The enzyme also catalyzes the stereospecific inversion of the C- 2’ hydroxyl of the nucleoside antibiotic, tubercidin to form 7-P-D-arabinofuranosyl-4- aminopyrrolo[2,3-d]pyrimidine. The nucleoside antibiotic, sangivamycin, in which the C- 5 hydrogen is replaced with a carboxamide group, is not a substrate. On the basis of the single and double label experiments in vivo and the in vitro enzyme-catalyzed experi- ments, two mechanisms involving either a 3’-ketonucleoside intermediate or a radical cation are proposed to explain the observed data. 0 1989 Academic Press, Inc. 9-@-D-Arabinofuranosyladenine (ara- A)3 was first isolated from culture filtrates of Streptorwgces antibioticus (1). The ara-A i This work was supported in part by a research grant (AI-22296) from the National Institutes of Health. 3 Abbreviations used: ara-A, 9-8-Parabinofurano- syladenine; dCF, deoxycoformycin; 2’-CldCF, 2’-chlo- ro-2’-deoxycoformycin. has a broad spectrum of activity against DNA viruses in cell culture and is success- fully used in therapy of herpes simplex en- cephalitis, neonatal herpes, herpes zoster, and chronic myelogenous leukemia (2, 3). The arabinofuranosyl moiety has also been identified in naturally occurring nucleo- sides, spongothymidine and spongouri- dine, which have been isolated from CQQ- tethia crypta (4,5). Several pathways have been proposed for the biosynthesis of ara- 363 0003-9861/89 33.00 Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form resewed.