Isolation, partial purification and characterization of an N-acetyltransferase from Streptomyces akiyoshiensis L-138

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Rodriguez-Juarez, Rocio C.
University of Lethbridge. Faculty of Arts and Science
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Lethbridge, Alta. : University of Lethbridge, Faculty of Arts and Science, 2003
A novel N-acetyltranferase (NAT) with high specificity for L-dopa was partially purified and characterized during this study. Streptomyces akiyoshiensis NAT was isolated from liquid cultures and the cell free extract was partially purified via two chromatographic approaches. The purest enzyme preparation (354.4-fold) was obtained through a combination of affinity (Affi-gel blue) and gel filtration chromatography. This preparation could not be stored due to poor stability. An alternate approach using affinity and anion-exchange column chromatogrpahy (macro-prep DEAE) provided a 125-fold purification. A second macro-prep DEAE showed the enrichment of a band at around 14.4 kDa. The enzyme activity was strongly inhibited by the presence of Cu2+ and Hg2+ salts suggesting the presence of critical histidine or cysteine redidues. In addition, NaC1 plays an important role as a stabilizing agent and/or slight activator of this enzyme. The Km and Vmax values for L-dopa determined at 30degreesC were 5.39 x 10-2 mM and 2.19 x 10-5 mM's, respectively. At 37 degreesC these kinetic constants were 0.11 mM and 2.57 x 10-4 mM's, respectively. The Km and Vmax values for AcCoA determined at 30 degreesC were 0.42 mM and 4.32 x 10-4 mM's, respectively. At 37 degreesC these values were 0.58 mM and 1.15 x 10-4 mM's, respectively. The optimal temperature and pH were 43 degreesC and 8.0 respectively. S. akiyoshiensis NAT acetylated arylalkylamine substrates but not arylamine substrates to any significant extent. These findings suggest that this enzyme but not arylamine subsrates to any significant extent. These findings suggest that this enzyme may be closely related to arylalkylamine-N-acetyltranferases (AANATs), however the certification of this postulate requires the knowledge of the amino acid sequence and ultimately 3D structure of S. akiyoshiensis NAT.
xvii, 128 leaves : ill. ; 28 cm.
Dissertations, Academic , Streptomyces , Acetyltransferases -- Purification