Preparation and properties of immobilized flavokinase

Merrill, Alfred H.; McCormick, Donald B.

doi:10.1002/bit.260210909

Cited by 10 publications

(8 citation statements)

References 15 publications

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“…This was shown in vitro by analyzing the enzymatic activity of the purified gene product RibC and also in vivo by complementation experiments using a ribCdefective B. subtilis strain. Flavokinases/FAD synthetases have been purified from several sources (2,26,30,37). In Corynebacterium ammoniagenes (RibF), B. subtilis (RibC), and E. coli (RibF), the flavokinase/FAD synthetase activities are provided by a single, bifunctional polypeptide chain (25, 26; K. Kitatsuji, S. Ishino, S. Teshiba, and M. Arimoto, Process for producing flavine mononucleotides.…”

Section: Discussionmentioning

confidence: 99%

The Bifunctional Flavokinase/Flavin Adenine Dinucleotide Synthetase from Streptomyces davawensis Produces Inactive Flavin Cofactors and Is Not Involved in Resistance to the Antibiotic Roseoflavin

et al. 2008

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Streptomyces davawensis synthesizes the antibiotic roseoflavin, one of the few known natural riboflavin analogs, and is roseoflavin resistant. It is thought that the endogenous flavokinase (EC 2.7.1.26)/flavin adenine dinucleotide (FAD) synthetase (EC 2.7.7.2) activities of roseoflavin-sensitive organisms are responsible for the antibiotic effect of roseoflavin, producing the inactive cofactors roseoflavin-5-monophosphate (RoFMN) and roseoflavin adenine dinucleotide (RoFAD) from roseoflavin. To confirm this, the FAD-dependent Sus scrofa D-amino acid oxidase (EC 1.4.3.3) was tested with RoFAD as a cofactor and found to be inactive. It was hypothesized that a flavokinase/FAD synthetase (RibC) highly specific for riboflavin may be present in S. davawensis, which would not allow the formation of toxic RoFMN/RoFAD. The gene ribC from S. davawensis was cloned. RibC from S. davawensis was overproduced in Escherichia coli and purified. ). Both RibC enzymes synthesized RoFAD and RoFMN. The functional expression of S. davawensis ribC did not confer roseoflavin resistance to a ribC-defective B. subtilis strain.Riboflavin (vitamin B 2 ) analogs have the potential to serve as basic structures for the development of novel anti-infectives (3). Consequently, both the mode of action of riboflavin analogs and the mechanism of resistance to these compounds are of substantial interest. Only very few natural riboflavin analogs are known (24). Moreover, the only known organism to produce a riboflavin analog with antibiotic activity is the grampositive bacterium Streptomyces davawensis (32).S. davawensis synthesizes the anti-vitamin 8-dimethyl-amino-8-demethyl-D-riboflavin, or roseoflavin ( Fig. 1), from riboflavin (27) via 8-amino-and 8-methylamino-8-demethyl-D-riboflavin (22). Roseoflavin is toxic to gram-positive but also to gramnegative bacteria if the compound is able to enter the cell (16). The molecular basis for roseoflavin toxicity is not clear. In all organisms, riboflavin serves as the direct precursor for the cofactors (ribo)flavin-5Ј-monophosphate (FMN) and flavin adenine dinucleotide (FAD) (12). FAD and (to a lesser extent) FMN are active components of flavoproteins, being involved in a wide range of redox and other reactions (15). FMN is synthesized from riboflavin by flavokinase (EC 2.7.1.26), and FAD is produced from FMN by FAD synthetase (EC 2.7.7.2) (2). In microorganisms, both enzymatic activities seem to be present in sufficient amounts, since only traces of free riboflavin are detectable in the cytoplasm (11). Accordingly, cytoplasmic roseoflavin may quickly be converted to the corresponding FMN/ FAD analogs roseoflavin-5Ј-monophosphate (RoFMN) and roseoflavin adenine dinucleotide (RoFAD). The synthesis of these inactive cofactors may explain the antibiotic activity of roseoflavin ( Fig. 1) (31, 36).S. davawensis is roseoflavin resistant, and the mechanism of self-resistance could involve a flavokinase/FAD synthetase with a high substrate specificity for riboflavin. Such an enzyme would not accept roseoflavin as...

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Section: Discussionmentioning

confidence: 99%

The Bifunctional Flavokinase/Flavin Adenine Dinucleotide Synthetase from Streptomyces davawensis Produces Inactive Flavin Cofactors and Is Not Involved in Resistance to the Antibiotic Roseoflavin

et al. 2008

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“…We also have immobilized enzymes as a means for flow-through catalysis. Examples are with D-amino oxidase (Tu & McCormick, 1972) and flavokinase (Merrill & McCormick, 1979).…”

Section: Biochemically Specific ('Affinity') Absorbentsmentioning

confidence: 99%

Micronutrient cofactor research with extensions to applications

McCormick

2002

Nutr. Res. Rev.

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Following identification of essential micronutrients, there has been a continuum of research aimed at revealing their absorption, transport, utilization as cofactors, and excretion and secretion. Among those cases that have received our attention are vitamin B 6 , riboflavin, biotin, lipoate, ascorbate, and certain metal ions. Circulatory transport and cellular uptake of the water-soluble vitamins exhibit relative specificity and facilitated mechanisms at physiological concentrations. Isolation of enzymes and metabolites from micro-organisms and mammals has provided information on pathways involved in cofactor formation and metabolism. Kinases catalysing phosphorylation of B 6 and riboflavin have a preference for Zn 2+ in stereospecific chelates with adenosine triphosphate. The synthetase for flavin adenine dinucleotide prefers Mg 2+ . The flavin mononucleotidedependent oxidase that converts the 5Ј-phosphates of pyridoxine and of pyridoxamine to pyridoxal phosphate is a connection between B 6 and riboflavin and is a primary control point for conversion of B 6 to its coenzyme. Sequencing and cloning of a side-chain oxidase for riboflavin was achieved. Details on binding and function have been delineated for some cofactor systems, especially in several flavoproteins. There is both photochemical oxidation and oxidative catabolism of B 6 and riboflavin. Both biotin and lipoate undergo oxidation of their acid side chains with redox cleavage of the rings. Applications from our findings include the development of affinity absorbents, enhanced drug delivery, delineation of residues in biopolymer modification, pathogen photoinactivation in blood components, and input into human dietary recommendations. Ongoing and future research in the cofactor arena can be expected to add to this panoply. At the molecular level, the way in which the same cofactor can participate in diverse catalytic reactions resides in interactions with surrounding enzyme structures that must be determined case by case. At the level of human intake, more knowledge is desirable for making micronutrient recommendations based on biochemical indicators, especially for the span between infancy and adulthood.

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“…<1> [7]) [7] P ADP + 10-(d-allo)flavin 5'-phosphate S ATP + 10-(l-arabo)flavin <1> (<1>, 25% of the activity with riboflavin [7]) (Reversibility: ? <1> [7]) [ [8]; <1>, as active as riboflavin [10]) (Reversibility: ? <1, 3, 5, 6> [8,9,12,13]) [8,9,10,12,13] P ADP + 5-deazariboflavin 5'-phosphate S ATP + 5-deazariboflavin 6 <5> (Reversibility: ?…”

Section: Reaction and Specificitymentioning

confidence: 99%

“…<1> [7]) [ [8]; <1>, as active as riboflavin [10]) (Reversibility: ? <1, 3, 5, 6> [8,9,12,13]) [8,9,10,12,13] P ADP + 5-deazariboflavin 5'-phosphate S ATP + 5-deazariboflavin 6 <5> (Reversibility: ? <5> [12]) [12] P ADP + 5-deazariboflavin 5'-phosphate S ATP + 5-methyl-5-deazariboflavin <5> (Reversibility: ?…”

Section: Reaction and Specificitymentioning

confidence: 99%

“…Temperature stability 25 <1> (<1>, enzyme immobilized by amide linkage to w aminoalkyl-agarose-beads has a half-life of three weeks [10]) [10] General stability information <1>, 50% of the activity is lost on freezing, but the stability of the enzyme is not greatly affected by the period of freezing [6] <1>, enzyme immobilized by amide linkage to w aminoalkyl-agarose-beads has a half-life of three weeks at 25 C [10] <1>, enzyme is inactivated by freezing and thawing unless both riboflavin and 20% glycerol are added [7] <3>, loss of activity during repeated freezing and thawing [9] Storage stability <1>, 3 C, MOPS buffer, activity decreases to approximately 35, 30 and 13% of the initial level after 24, 48, and 96 h, respectively [6] <1>, 4 C, 50% loss of activity after 2 d when the enzyme is stored in buffer alone, complete protection by 0.01 mM riboflavin [7] <1>, purified enzyme is unstable in dilute solution and can not be stored at -20 C [1]…”

Section: Reaction and Specificitymentioning

confidence: 99%

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Riboflavin kinase

Springer Handbook of Enzymes

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Preparation and properties of immobilized flavokinase

Cited by 10 publications

References 15 publications

The Bifunctional Flavokinase/Flavin Adenine Dinucleotide Synthetase from Streptomyces davawensis Produces Inactive Flavin Cofactors and Is Not Involved in Resistance to the Antibiotic Roseoflavin

The Bifunctional Flavokinase/Flavin Adenine Dinucleotide Synthetase from Streptomyces davawensis Produces Inactive Flavin Cofactors and Is Not Involved in Resistance to the Antibiotic Roseoflavin

Micronutrient cofactor research with extensions to applications

Riboflavin kinase

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