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Title: Reconstitution of {beta}-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase

CYP175A1 is a thermostable P450 Monooxygenase from Thermus thermophilus HB27, demonstrating in vivo activity towards {beta}-carotene. Activity of CYP175A1 was reconstituted in vitro using artificial electron transport proteins. First results were obtained in the mixture with a crude Escherichia coli cell extract at 37 {sup o}C. In this system, {beta}-carotene was hydroxylated to {beta}-cryptoxanthin. The result indicated the presence of electron transport enzymes among the E. coli proteins, which are suitable for CYP175A1. However, upon in vitro reconstitution of CYP175A1 activity with purified recombinant flavodoxin and flavodoxin reductase from E. coli, only very low {beta}-cryptoxanthin production was observed. Remarkably, with another artificial electron transport system, putidaredoxin and putidaredoxin reductase from Pseudomonas putida, purified CYP175A1 enzyme hydroxylated {beta}-carotene at 3- and also 3'-positions, resulting in {beta}-cryptoxanthin and zeaxanthin. Under the optimal reaction conditions, the turnover rate of the enzyme reached 0.23 nmol {beta}-cryptoxanthin produced per nmol P450 per min.
Authors:
 [1] ;  [2] ;  [1] ;  [3]
  1. Institute of Technical Biochemistry, University of Stuttgart, Stuttgart 70569 (Germany)
  2. Dr. Margarete Fischer-Bosch-Institut fuer Klinische Pharmakologie, Stuttgart 70376 (Germany)
  3. Institute of Technical Biochemistry, University of Stuttgart, Stuttgart 70569 (Germany). E-mail: itbvkha@po.uni-stuttgart.de
Publication Date:
OSTI Identifier:
20795860
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 339; Journal Issue: 1; Other Information: DOI: 10.1016/j.bbrc.2005.11.023; PII: S0006-291X(05)02534-9; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CAROTENOIDS; ESCHERICHIA COLI; HYDROXYLASES; IN VITRO; IN VIVO; PSEUDOMONAS