Biosynthesis of 9-beta-D-arabinofuranosyladenine: hydrogen exchange at C-2' and oxygen exchange at C-3' of adenosine.
The data presented here describe new findings related to the bioconversion of adenosine to 9-beta-D-arabinofuranosyladenine (ara-A) by Streptomyces antibioticus by in vivo investigations and with a partially purified enzyme. First, in double label in vivo experiments with [2'-18O]- and [U-14C]adenosine, the 18O:14C ratio of the ara-A isolated does not change appreciably, indicating a stereospecific inversion of the C-2' hydroxyl of adenosine to ara-A with retention of the 18O at C-2'. In experiments with [3'-18O]- and [U-14C]-adenosine, [U-14C]ara-A was isolated; however, the 18O at C-3' is below detection. The adenosine isolated from the RNA from both double label experiments has essentially the same ratio of 18O:14C. 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 experiment 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 3H2O. Third, the release of 3H as 3H2O from [2'-3H]adenosine was confirmed by incubations of the enzyme with 3H2O 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 10(-4) 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-beta-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 experiments, two mechanisms involving either a 3'-ketonucleoside intermediate or a radical cation are proposed to explain the observed data.
Suhadolnik
R J
RJ
Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Pornbanlualap
S
S
Wu
J M
JM
Baker
D C
DC
Hebbler
A K
AK
D000241:Adenosine / Q000378:metabolism; D000243:Adenosine Deaminase / Q000096:biosynthesis; D002854:Chromatography, Paper; D008401:Gas Chromatography-Mass Spectrometry; D006859:Hydrogen / Q000378:metabolism*; D007553:Isotope Labeling; D007700:Kinetics; D013058:Mass Spectrometry; D010100:Oxygen / Q000378:metabolism*; D012588:Scintillation Counting; D013302:Streptomyces / Q000378:metabolism*; D013303:Streptomyces antibioticus / Q000201:enzymology / Q000378:metabolism*; D013379:Substrate Specificity; D014740:Vidarabine / Q000096:biosynthesis*; D014867:Water / Q000378:metabolism
D014867:Water; D006859:Hydrogen; D000243:Adenosine Deaminase; D014740:Vidarabine; D000241:Adenosine; D010100:Oxygen