Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have placed the PHR1 gene of Saccharomyces cerevisiae under the transcriptional and translational control of the tac expression cartridge. Under inducing conditions Escherichia coli cells harboring plasmids carrying this construct accumulate approximately 8% of total cellular protein as the Phr1 photolyase. Using a strain devoid of E. coli photolyase activity, we have obtained milligram quantities of the yeast enzyme at greater than 95% purity and have characterized the enzyme. Phr1 photolyase is a monomer in solution with an Mr of 60,000, has a turnover number of 0.7 dimers min-1 molecule-1 in vitro, exhibits absorbance maxima at lambda = 277 and 377 nm, and has a fluorescence excitation maximum at 390 nm and an emission maximum at 475 nm. The near UV absorbance peak is shown to reflect the contributions of two intrinsic chromophores which are noncovalently bound to the enzyme. Spectroscopic, fluorescence, and thin layer chromatographic studies indicate that one of these chromophores is 1,5-reduced FAD rather than 4a,5-reduced FAD as previously proposed (Iwatsuki, N., Joe, C. O., and Werbin, H. (1980) Biochemistry 19, 1172-1176), while the other chromophore has properties similar to the second chromophore of E. coli photolyase. The fact that yeast and E. coli photolyases are similar both with respect to amino acid sequence and chromophore composition provides strong evidence that the enzymes share a common action mechanism which may also be utilized by photolyases from other organisms throughout the phylogenetic tree.
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PMID:Purification of the yeast PHR1 photolyase from an Escherichia coli overproducing strain and characterization of the intrinsic chromophores of the enzyme. 331 99

DNA photolyases use two noncovalently bound chromophores to catalyze photoreactivation, the blue light-dependent repair of DNA that has been damaged by ultraviolet light. FAD is the catalytic chromophore for all photolyases and is essential for photoreactivation. The identity of the second chromophore is often 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO). Under standard light conditions, the second chromophore is considered nonessential for photoreactivation because DNA photolyase bound to only FAD is sufficient to catalyze the repair of UV-damaged DNA. phr1 is a photoreactivation-deficient strain of Chlamydomonas. In this work, the PHR1 gene of Chlamydomonas was cloned through molecular mapping and shown to encode a protein similar to known FO synthases. Additional results revealed that the phr1 strain was deficient in an FO-like molecule and that this deficiency, as well as the phr1 photoreactivation deficiency, could be rescued by transformation with DNA constructs containing the PHR1 gene. Furthermore, expression of a PHR1 cDNA in Escherichia coli produced a protein that generated a molecule with characteristics similar to FO. Together, these results indicate that the Chlamydomonas PHR1 gene encodes an FO synthase and that optimal photoreactivation in Chlamydomonas requires FO, a molecule known to serve as a second chromophore for DNA photolyases.
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PMID:Critical role of 7,8-didemethyl-8-hydroxy-5-deazariboflavin for photoreactivation in Chlamydomonas reinhardtii. 2069 62