Parry, Ronald J.2009-06-042009-06-041998Jiang, Yijia. "Investigations of neplanocin a reductase, an enzyme involved in aristeromycin biosynthesis." (1998) Diss., Rice University. <a href="https://hdl.handle.net/1911/19273">https://hdl.handle.net/1911/19273</a>.https://hdl.handle.net/1911/19273The nucleoside antibiotics aristeromycin and neplanocin A are two naturally occurring carbocyclic analogs of adenosine produced by Streptomyces citricolor. Partial purification of the crude cell-free extracts of S. citricolor by DEAE chromatography, ammonium sulfate fractionation, and chromatography on phenyl agarose yielded a cell-free system that converted neplanocin A to aristeromycin as judged by HPLC and NMR analysis. The production of aristeromycin was dependent upon the addition of NADPH to the incubation mixture and $\rm NAD\sp+$ was found to increase the production of aristeromycin at very low concentration when combined with NADPH. The apparent $\rm K\sb{m}\sp{s}$ of neplanocin A and NADPH were determined to be 15.4 $\rm\mu M$ and 11.1 $\rm\mu M$ for neplanocin A reductase using phenyl agarose purified enzyme. The native molecular weight of the reductase is around 50 KDa when measured by gel filtration chromatography and the pl of the enzyme is 4.8. The mechanism of the reduction reaction was explored by incubation of neplanocin A with isotopically labeled NADPH and partially purified reductase, and by incubation of the enzyme with NADPH in a deuterated buffer. The mechanism of the double bond reduction was further studied with the substrate analogs $4\sp\prime$-deshydroxymethyl- and $3\sp\prime$-deoxy-neplanocin A, and by isotope exchange experiments with aristeromycin and neplanocin A in deuterated buffer containing $\rm NAD\sp+.$ The results of these investigations will be presented in this thesis.127 p.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.BiochemistryOrganic chemistryThesisTHESIS CHEM. 1998 JIANG