Enzyme Catalysis via Control of Activation Entropy: Site-directed Mutagenesis of 6,7-Dimethyl-8-ribityllumazine Synthase

Fischer, Markus; Haase, Ilka; Kis, Klaus; Meining, Winfried; Ladenstein, Rudolf; Cushman, Mary; Schramek, Nicholas; Huber, Robert; Bacher, Adelbert

doi:10.1016/s0022-2836(02)01473-0

Cited by 38 publications

(45 citation statements)

References 32 publications

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“…The full length of the ribE gene, encoding lumazine synthase, was amplified and sequenced by using forward (ribE-FW; GCA TTT AGT GGG TGC ATG ATC) and reverse (ribE-RV; GGA ACT GGT ATT CAA CAT CAG CG) primers, and the sequence was compared to the wild-type J53 sequence (GenBank accession number AICK00000000). Nonsynonymous mutations involving active-site residues and loss-of-function mutations were considered significant (30,31).…”

Section: Methodsmentioning

confidence: 99%

Plasmid-Mediated OqxAB Is an Important Mechanism for Nitrofurantoin Resistance in Escherichia coli

et al. 2016

Antimicrob Agents Chemother

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Increasing consumption of nitrofurantoin (NIT) for treatment of acute uncomplicated urinary tract infections (UTI) highlights the need to monitor emerging NIT resistance mechanisms. This study investigated the molecular epidemiology of the multidrugresistant efflux gene oqxAB and its contribution to nitrofurantoin resistance by using Escherichia coli isolates originating from patients with UTI (n ‫؍‬ 205; collected in 2004 to 2013) and food-producing animals (n ‫؍‬ 136; collected in 2012 to 2013) in Hong Kong. The oqxAB gene was highly prevalent among NIT-intermediate (11.5% to 45.5%) and -resistant (39.2% to 65.5%) isolates but rare (0% to 1.7%) among NIT-susceptible (NIT-S) isolates. In our isolates, the oqxAB gene was associated with IS26 and was carried by plasmids of diverse replicon types. Multilocus sequence typing revealed that the clones of oqxAB-positive E. coli were diverse. The combination of oqxAB and nfsA mutations was found to be sufficient for high-level NIT resistance. Curing of oqxAB-carrying plasmids from 20 NIT-intermediate/resistant UTI isolates markedly reduced the geometric mean MIC of NIT from 168.9 g/ml to 34.3 g/ml. In the plasmid-cured variants, 20% (1/5) of isolates with nfsA mutations were NIT-S, while 80% (12/15) of isolates without nfsA mutations were NIT-S (P ‫؍‬ 0.015). The presence of plasmid-based oqxAB increased the mutation prevention concentration of NIT from 128 g/ml to 256 g/ml and facilitated the development of clinically important levels of nitrofurantoin resistance. In conclusion, plasmid-mediated oqxAB is an important nitrofurantoin resistance mechanism. There is a great need to monitor the dissemination of this transferable multidrug-resistant efflux pump.

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Section: Methodsmentioning

confidence: 99%

Plasmid-Mediated OqxAB Is an Important Mechanism for Nitrofurantoin Resistance in Escherichia coli

et al. 2016

Antimicrob Agents Chemother

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“…Moreover, the activation energy of the noncatalyzed reaction is lower than that of the enzymecatalyzed reaction, at least in the case of the enzymes from eubacteria and spinach (118,225).…”

Section: 4-dihydroxy-2-butanone 4-phosphate Synthasementioning

confidence: 99%

“…The topologically equivalent active sites (5 in the case of the pentameric enzymes and 60 in the case of the icosahedral enzymes) are all located at the interfaces between adjacent subunits in the pentamer motif. The structural complexity of some of these proteins is in surprising contrast with the absence of any amino acid residues that can individually be of major importance for the enzyme-catalyzed reaction (112,118). In Bacillaceae, lumazine synthase and RF synthase form a complex comprising an icosahedral capsid of 60 lumazine synthase subunits and a core of 3 RF synthase subunits (see below for more details) (12,19).…”

Section: 4-dihydroxy-2-butanone 4-phosphate Synthasementioning

confidence: 99%

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

Abbas

Sibirny

2011

Microbiol Mol Biol Rev

316

272

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SUMMARY Riboflavin [7,8-dimethyl-10-(1′- d -ribityl)isoalloxazine, vitamin B 2 ] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pteridine intermediates. Flavin nucleotides are synthesized in two consecutive reactions from riboflavin. Some microorganisms and all animal cells are capable of riboflavin uptake, whereas many microorganisms have distinct systems for riboflavin excretion to the medium. Regulation of riboflavin synthesis in bacteria occurs by repression at the transcriptional level by flavin mononucleotide, which binds to nascent noncoding mRNA and blocks further transcription (named the riboswitch). In flavinogenic molds, riboflavin overproduction starts at the stationary phase and is accompanied by derepression of enzymes involved in riboflavin synthesis, sporulation, and mycelial lysis. In flavinogenic yeasts, transcriptional repression of riboflavin synthesis is exerted by iron ions and not by flavins. The putative transcription factor encoded by SEF1 is somehow involved in this regulation. Most commercial riboflavin is currently produced or was produced earlier by microbial synthesis using special selected strains of Bacillus subtilis , Ashbya gossypii , and Candida famata . Whereas earlier RF overproducers were isolated by classical selection, current producers of riboflavin and flavin nucleotides have been developed using modern approaches of metabolic engineering that involve overexpression of structural and regulatory genes of the RF biosynthetic pathway as well as genes involved in the overproduction of the purine precursor of riboflavin, GTP.

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“…In fact, all eight carbon atoms of the xylene moiety are derived from the product of the enzyme. In the biosynthetic pathway, 3,4-dihydroxy-2-butanone 4-phosphate is condensed with 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione derived from GTP (14,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). The reaction product, 6,7-dimethyl-8-ribityllumazine, undergoes a highly unusual dismutation catalyzed by riboflavin synthase, which involves the exchange of a 4-carbon fragment between two identical substrate molecules.…”

mentioning

confidence: 99%

Structure of 3,4-Dihydroxy-2-butanone 4-Phosphate Synthase from Methanococcus jannaschii in Complex with Divalent Metal Ions and the Substrate Ribulose 5-Phosphate

Steinbacher

Schiffmann

Richter

et al. 2003

Journal of Biological Chemistry

Self Cite

107

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Skeletal rearrangements of carbohydrates are crucial for many biosynthetic pathways. In riboflavin biosynthesis ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions. Here, we present the crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal center presumably consisting of non-catalytic zinc and calcium ions at 1.7-Å resolution. The carbonyl group (O2) and two out of three free hydroxyl groups (OH3 and OH4) of the substrate are metal-coordinated. We correlate previous mutational studies on this enzyme with the present structural results. Residues of the first coordination sphere involved in metal binding are indispensable for catalytic activity. Only Glu-185 of the second coordination sphere cannot be replaced without complete loss of activity. It contacts the C3 hydrogen atom directly and probably initiates enediol formation in concert with both metal ions to start the reaction sequence. Mechanistic similarities to Rubisco acting on the similar substrate ribulose 1,5-diphosphate in carbon dioxide fixation as well as other carbohydrate (reducto-) isomerases are discussed.Riboflavin (vitamin B 2 ) is biosynthesized in plants and numerous microorganisms but not in animals, which depend on nutritional sources. Its derivatives, flavin mononucleotide (FMN) and flavinadenine dinucleotide (FAD), are indispensable in all cells where they serve a variety of redox reactions (1). They have also been shown to serve a variety of other functions in cells such as DNA photorepair (2), light sensing (3, 4), and bioluminescence (5-8) and in reactions without net-redox change (9).Gram-negative bacteria are absolutely dependent on endogenous synthesis of riboflavin, because they are devoid of an uptake system for flavins or flavocoenzymes. Hence, riboflavindeficient mutants e.g. of Escherichia coli and Salmonella sp. require extremely high concentrations of exogenous riboflavin for growth. The same is true for yeasts such as Saccharomyces cerevisiae and Candida guilliermondii (for review see Refs. 1 and 10 -13). Thus, these organisms should be vulnerable to inhibitors of the riboflavin biosynthesis, which could therefore qualify as novel anti-infective agents. The absence of the riboflavin pathway in the human host appears advantageous in this context, because host/parasite selectivity of inhibitory agents would not constitute a problem. The mechanistic and structural analysis of the riboflavin pathway could serve as the basis for the rational design of riboflavin pathway inhibitors.3,4-Dihydroxy-2-butanone 4-phosphate synthase supplies the building blocks for the assembly of the xylene ring of the vitamin (14 -17). In fact, all eight carbon atoms of the xylene moiety are derived from the product of the enzyme. In the biosynthetic pathway, 3,4-dihydroxy-2-butanone 4-phosphate is condensed with 5-amino-6-ribitylamino-2,4(1H,3H)-...

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Enzyme Catalysis via Control of Activation Entropy: Site-directed Mutagenesis of 6,7-Dimethyl-8-ribityllumazine Synthase

Cited by 38 publications

References 32 publications

Plasmid-Mediated OqxAB Is an Important Mechanism for Nitrofurantoin Resistance in Escherichia coli

Plasmid-Mediated OqxAB Is an Important Mechanism for Nitrofurantoin Resistance in Escherichia coli

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

Structure of 3,4-Dihydroxy-2-butanone 4-Phosphate Synthase from Methanococcus jannaschii in Complex with Divalent Metal Ions and the Substrate Ribulose 5-Phosphate

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