Presteady State Kinetic Analysis of Riboflavin Synthase

Abstract

Riboflavin synthase catalyzes a mechanistically complex dismutation affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione from 6,7-dimethyl-8-ribityllumazine. The kinetics of the enzyme from Escherichia coli were studied under single turnover conditions. Stopped flow as well as quenched flow experiments documented the transient formation of a pentacyclic reaction intermediate. No other transient species were sufficiently populated to allow detection. The data are best described by a sequence of one second order and one first order reaction.Riboflavin synthase catalyzes a complex dismutation affording riboflavin (8) and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (9) from 6,7-dimethyl-8-ribityllumazine (1) (1). The enzyme-catalyzed dismutation can be described as an exchange of a 4-carbon unit between one substrate molecule acting as donor and a second substrate molecule acting as acceptor (typically, however, dismutation reactions involve the exchange of simple particles, e.g. hydride anions) (1, 2). The regiochemical features of the reaction require an antiparallel arrangement of the two identical substrate molecules at the active site of the enzyme (3-5).Riboflavin synthases of Bacillus subtilis and Escherichia coli are homotrimers of 23.4-kDa subunits (6, 7). Each subunit folds into two closely similar domains (7,8). The homotrimeric enzymes have six substrate-binding sites (one binding site each on each N-terminal and C-terminal domain, respectively, of each subunit) (9 -11). Surprisingly, the crystal structure of riboflavin synthase from E. coli shows an inherently asymmetric molecule where only a single N-terminal domain and a single C-terminal domain are appropriately oriented for catalysis of a two-substrate reaction (8).Recently, we reported the formation of the pentacyclic lumazine dimer 6 from 6,7-dimethyl-8-ribityllumazine by the S41A mutant of E. coli riboflavin synthase (12). The native enzyme can generate 6,7-dimethyl-8-ribityllumazine (backward reaction) as well as a mixture of riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (forward reaction) from that adduct, which fulfills the criteria for a kinetically competent reaction intermediate. The hypothetical reaction mechanism shown in Fig. 1 combines the earlier mechanistic suggestions of Rowan and Wood (13) and Beach and Plaut (14) with the more recent findings.This paper describes presteady state kinetic studies on this mechanistically complex enzyme-catalyzed reaction. and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione were synthesized by published procedures (15). A recombinant strain of E. coli engineered for overexpression of the ribC gene specifying riboflavin synthase of E. coli has been described elsewhere (16). EXPERIMENTAL PROCEDURES MaterialsProtein Purification-All procedures were performed at 4°C unless otherwise stated. Frozen cell mass (5 g) was thawed in 25 ml of 50 mM Tris hydrochloride, pH 7.2, containing 0.5 mM EDTA and 0.5 mM dithiothreitol (buffer A). The suspension was subjecte...

Structures and reaction mechanisms of riboflavin synthases of eubacterial and archaeal origin

“…In these experiments, no other transient species were sufficiently populated to allow detection [31,32].…”

Evolution of Vitamin B2 Biosynthesis. A Novel Class of Riboflavin Synthase in Archaea

“…[6][7][8][9][10][11] The final step in the biosynthesis of the vitamin involves the dismutation of 6,7-dimethyl-8-ribityllumazine (5) catalyzed by riboflavin synthase. [12][13][14][15] 0022-2836/$ -see front matter q 2004 Elsevier Ltd. All rights reserved. † This paper is dedicated to the memory of Dr Justus Henatsch, Europa Fachhochschule Fresenius Idstein.…”

Biocatalytic Strategies towards [4+2] Cycloadditions

“…Riboflavin synthase catalyses an intermolecular cyclisation reaction that is part of primary metabolism and should therefore be old on an evolutionary time scale. This reaction leads to the biosynthesis of the essential cofactor riboflavin via pentacyclic intermediate 29 (Figure A) . Several mechanisms have been proposed for the riboflavin synthase‐catalysed dimerisation of 6,7‐dimethyllumazine 30 but DFT calculations in water have ruled out most of them .…”