tailieunhanh - Báo cáo khoa học: Biosynthesis of riboflavin Screening for an improved GTP cyclohydrolase II mutant

GTP cyclohydrolase II catalyzes the first dedicated step in the biosynthesis of riboflavin and appears to be a limiting factor for the production of the vitamin by recombinantBacillus subtilisoverproducer strains. Using error-prone PCR amplification, we generated a library of theB. subtilis ribA gene selectively mutated in the GTP cyclohydrolase II domain. | ỊFEBS Journal Biosynthesis of riboflavin Screening for an improved GTP cyclohydrolase II mutant Martin Lehmann1 Simone Degen1 Hans-Peter Hohmann1 Markus Wyss1 Adelbert Bacher2 and Nicholas Schramek2 1 DSM NutritionalProducts Ltd. Basel Switzerland 2 Lehrstuhl fur Biochemie Technische Universitat Munchen Lichtenbergstr Garching Germany Keywords biotechnology directed evolution GTP cyclohydrolase riboflavin biosynthesis vitamin B2 production Correspondence N. Schramek Lehrstuhl fur Biochemie Technische Universitat Munchen Lichtenbergstr. 4 D-85747 Garching Germany Tel 49 089 289 13336 Fax 49 089 289 13363 E-Mail Received 16 March 2009 Revised 24 May 2009 accepted 28 May 2009 doi GTP cyclohydrolase II catalyzes the first dedicated step in the biosynthesis of riboflavin and appears to be a limiting factor for the production of the vitamin by recombinant Bacillus subtilis overproducer strains. Using error-prone PCR amplification we generated a library of the B. subtilis ribA gene selectively mutated in the GTP cyclohydrolase II domain. The ratio of the GTP cyclohydrolase II to 3 4-dihydroxy-2-butanone synthase activities of the mutant proteins was measured. A mutant designated Construct E carrying seven point mutations showed a two-fold increase in GTP cyclohydrolase II activity and a four-fold increase in the Km value with GTP as the substrate. Using the analog 2-amino-5-formylamino-6-ribosylamino-4 3H -pyrimidinone 5 -triphosphate as the substrate the mutant showed a rate enhancement by a factor of about two and an increase in the Km value by a factor of about 5. A series of UV absorption spectra obtained in stopped-flow experiments using the wild-type and mutant enzymes revealed isosbestic points indicative of apparently perfect reactions which were similar to the findings obtained with GTP cyclohydrolase II of Escherichia coli. Initial burst velocities obtained for the mutant and wild-type proteins were .