Purification and characterization of UDP-glucose : tetrahydrobiopterin glucosyltransferase from Synechococcus sp PCC 7942

Tetrahydrobiopterin (BH4)-glucoside was identified from Synechococcus sp. P CC 7942 by HPLC analysis and the enzymatic activity of a glycosyltransferas e producing the compound from UDP-glucose and BH4. The novel enzyme, named UDP-glucose:BH4 glucosyltransferase, has been purified 846-fold from the cy tosolic fraction of Synechococcus sp. PCC 7942 to apparent homogeneity on S DS-PAGE. The native enzyme exists as a monomer having a molecular mass of 3 9.2 kDa on SDS-PAGE. The enzyme was active over a broad range of pH from 6. 5 to 10.5 but most active at pH 10.0. The enzyme required Mn2+ for maximal activity. Optimum temperature was 42 degreesC. Apparent K-m values for BH4 and UDP-glucose were determined as 4.3 muM and 188 muM, respectively, and V -max values were 16.1 and 15.1 pmol min(-1) mg(-1), respectively. The N-ter minal amino acid sequence was Thr-Ala-His-Ar g-Phe-Lys-Phe-Val-Ser-Thr shar ing high homology with the predicted N-terminal sequence of an unidentified open reading frame slr1166 determined in the genome of Synechocystis sp. P CC 6803, which is known to produce a pteridine glycoside cyanopterin. (C) 2 000 Elsevier Science B.V. All rights reserved.