Tài liệu Báo cáo khoa học: Biosynthesis of riboflavin Screening for an improved GTP cyclohydrolase II

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 Nutritional Products Ltd., Basel, Switzerland

2 Lehrstuhl fu¨r Biochemie, Technische Universita¨t Mu¨nchen, Lichtenbergstr, Garching, Germany

Introduction

More than 3000 metric tons of vitamin B2 (riboflavin;

6) are produced per year for use in human nutrition,

animal husbandry and as a food colorant. In recent

years, efficient fermentation processes have replaced

chemical synthesis for manufacturing the vitamin [1,2].

The biosynthetic pathway of riboflavin has been

studied in considerable detail [3–6]. Briefly, GTP is

converted into 2,5-diamino-6-ribosylamino-4(3H)-pyri￾midinone 5¢-phosphate (2) by the catalytic action of

GTP cyclohydrolase II (Fig. 1) [7]. The product is

transformed into 5-amino-6-ribitylamino-2,4(1H,3H)-

pyrimidinedione (3) by a sequence of side-chain reduc￾tion, deamination and dephosphorylation. Condensa￾tion of 3 with 3,4-dihydroxy-2-butanone 4-phosphate

(4) results in the production of 6,7-dimethyl-8-ribityl￾lumazine (5) [8,9]. An unusual dismutation catalyzed

by riboflavin synthase converts the lumazine derivative

into an equimolar mixture of riboflavin (6) and the

pyrimidine 3 which is re-utilized by the lumazine

synthase [10–13]. With the exception of the elusive

phosphatase, all enzymes of the pathway have been

studied at least in some detail.

The enzymes of the riboflavin pathway are generally

characterized by low catalytic rates. This is not surprising

Keywords

biotechnology; directed evolution; GTP

cyclohydrolase; riboflavin biosynthesis;

vitamin B2 production

Correspondence

N. Schramek, Lehrstuhl fu¨r Biochemie,

Technische Universita¨t Mu¨nchen,

Lichtenbergstr. 4, D-85747 Garching,

Germany

Tel: +49 089 289 13336

Fax: +49 089 289 13363

E-Mail: [email protected]

(Received 16 March 2009, Revised 24 May

2009, accepted 28 May 2009)

doi:10.1111/j.1742-4658.2009.07118.x

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 cyclo￾hydrolase 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 simi￾lar to the findings obtained with GTP cyclohydrolase II of Escherichia coli.

Initial burst velocities obtained for the mutant and wild-type proteins were

similar. The data suggest that the mutations present in Construct E are

jointly conducive to the acceleration of a late step in the reaction trajec￾tory, most probably the release of product from the enzyme.

Abbreviation

DHB, 3,4-dihydroxy-2-butanone.

FEBS Journal 276 (2009) 4119–4129 ª 2009 The Authors Journal compilation ª 2009 FEBS 4119