Crystal Structure of an Archaeal Pentameric Riboflavin Synthase in Complex with a Substrate Analog Inhibitor

Abstract

Whereas eubacterial and eukaryotic riboflavin synthases form homotrimers, archaeal riboflavin synthases from Methanocaldococcus jannaschii and Methanothermobacter thermoautrophicus are homopentamers with sequence similarity to the 6,7-dimethyl-8-ribityllumazine synthase catalyzing the penultimate step in riboflavin biosynthesis. Recently it could be shown that the complex dismutation reaction catalyzed by the pentameric M. jannaschii riboflavin synthase generates riboflavin with the same regiochemistry as observed for trimeric riboflavin synthases. Here we present crystal structures of the pentameric riboflavin synthase from M. jannaschii and its complex with the substrate analog inhibitor, 6,7-dioxo-8-ribityllumazine. The complex structure shows five active sites located between adjacent monomers of the pentamer. Each active site can accommodate two substrate analog molecules in anti-parallel orientation. The topology of the two bound ligands at the active site is well in line with the known stereochemistry of a pentacyclic adduct of 6,7-dimethyl-8-ribityllumazine that has been shown to serve as a kinetically competent intermediate. The pentacyclic intermediates of trimeric and pentameric riboflavin synthases are diastereomers.Riboflavin (vitamin B 2 ) serves as the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), essential cofactors for several oxidoreductases that are indispensable in most living cells. The work on riboflavin biosynthesis in microorganisms has been covered extensively in recent reviews (1-4). Riboflavin is biosynthesized in plants, many bacteria, and in fungi but not in animals. Therefore, enzymes of this pathway have been proposed to be attractive targets for antimicrobial strategies (5-7).In the final steps of the biosynthetic pathway, lumazine synthase (LS) catalyzes the condensation of the pyrimidinedione (1) with 3,4-dihydroxy-2-butanone-4-phosphate (2) to release water, inorganic phosphate and 6,7-dimethyl-8-ribityllumazine (DMRL) 7 (8, 9), and riboflavin synthase (RS) catalyzes a dismutation of DMRL (3) affording riboflavin (4) and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (1) (Fig. 1); more specifically, that reaction involves the transfer of a four-carbon moiety between two DMRL molecules serving as donor and acceptor, respectively (10 -13). Both reactions are thermodynamically irreversible (9, 14) and can proceed in the absence of a catalyst (11,(15)(16)(17).LS from fungi are c 5 -symmetric homopentamers, whereas the enzymes from plants, Archaea, and most eubacteria studied are 532 symmetric capsids of 60 identical subunits, which are best described as dodecamers of pentamers. The subunit folding patterns of pentameric and icosahedral LS are similar. In Bacillaceae, lumazine synthase and riboflavin synthase form a complex comprising an icosahedral capsid of 60 lumazine synthase subunits and a core of three riboflavin synthase subunits; historically, these unusual enzyme complexes were designated heavy riboflavin synthase (18,19). Riboflavin syn...

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

“…In archaea, there is a fundamental difference in the stereochemistry of the pentacyclic intermediate involved in the dismutation of 6,7-dimethyl-8-ribityllumazine into RF and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione in the last step of RF biosynthesis (190). Notably, the active sites of the RF synthase from archaea have the same basic topology as that of lumazine synthase (362).…”

“…All the enzymes mentioned are homotrimers. However, RF synthases from archaea are homopentamers, with unrelated amino acid sequences (121,362).…”

Evolution of Vitamin B2Biosynthesis: 6,7-Dimethyl-8-Ribityllumazine Synthases ofBrucella

“…From this analysis, depicted in Fig. 5, a completely new picture emerges, showing that in addition to eubacterial type I lumazine synthases and archaeal lumazine synthases, this folding gave origin to at least two new evolutionarily related functions: the archaeal riboflavin synthases, which are lumazine synthaselike riboflavin synthases with no detectable lumazine synthase activity (13,15,45), and the eubacterial type II lumazine synthases.…”

“…The topologically equivalent active sites (5 in the case of the pentameric enzymes and 60 in the case of the icosahedral enzymes) are invariably located at interfaces between adjacent subunits in the pentamer moieties. Recently, it was found that pentameric riboflavin synthases of archaea are closely related to 6,7-dimethyl-8-ribityllumazine synthases (15,25,45).Brucellosis is a disease of humans and livestock that is caused by closely related Brucella species adapted to intracellular life within the cells of a variety of mammals; the main pathogenic species for domestic animals are Brucella abortus, Brucella melitensis, and Brucella suis. Goldbaum and coworkers have shown that an 18-kDa B.…”

“…The topologically equivalent active sites (5 in the case of the pentameric enzymes and 60 in the case of the icosahedral enzymes) are invariably located at interfaces between adjacent subunits in the pentamer moieties. Recently, it was found that pentameric riboflavin synthases of archaea are closely related to 6,7-dimethyl-8-ribityllumazine synthases (15,25,45).…”