Chorismate synthase of Neurospora crassa: A flavoprotein

Welch, G. Rickey; Cole, Kenneth W.; Gaertner, Frank

doi:10.1016/0003-9861(74)90276-8

Cited by 61 publications

(44 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1 (panel B, M98Kϩ) and allowed the exclusive expression of the full-length enzyme. The purified M98K mutant protein has a specific activity of 0.8 units/mg, similar to the value of 0.7 units/mg reported by Welch et al (7) for wild-type enzyme. Because the introduced mutation had no effect on the specific activity of the enzyme, the M98K mutant protein was used in all experiments throughout this study and is referred to as NcCS.…”

Section: Methodssupporting

confidence: 86%

“…UV-Visible Absorbance Spectrophotometry of NcCS-Purified recombinant NcCS was completely devoid of bound FMN (data not shown), indicating weak binding of the cofactor (7). A similar finding has been reported for the enzyme from E. coli (6) and Thermotoga maritima (18).…”

Section: Expression and Purification Of N Crassa Chorismate Synthasesupporting

confidence: 74%

“…On the other hand, chorismate synthase from fungal sources, i.e. Saccharomyces cerevisiae and Neurospora crassa, have been found to possess an additional intrinsic catalytic activity in that they can utilize NADPH to reduce the flavin cofactor (7,8). These enzymes have been termed bifunctional.…”

mentioning

confidence: 99%

“…So far bifunctionality appears to be restricted to chorismate synthases from fungal species, and the physiological implications of this exclusive occurrence in fungi are not known. Although the bifunctional chorismate synthase from N. crassa was among the first chorismate synthases studied (7), the low abundance of the protein has prevented detailed studies of its biochemical properties. The higher molecular mass of the N. crassa enzyme (by 5-8 kDa) as compared with monofunctional bacterial and plant enzymes led to the hypothesis that N. crassa CS contains an additional NADPH binding site (9).…”

mentioning

confidence: 99%

“…In these enzymes, NADPH-dependent reduction of flavin is up to 5 orders of magnitude faster in the presence of the substrate. In contrast to reactions in which redox equivalents are consumed stoichiometrically during substrate turnover, the FMN cofactor of chorismate synthase remains in its active reduced form during EPSP consumption (7,12). However, molecular oxygen inactivates the enzyme as it reacts with reduced flavin to yield the oxidized flavin and hydrogen peroxide.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Spectroscopic and Kinetic Characterization of the Bifunctional Chorismate Synthase from Neurospora crassa

Kitzing

Macheroux

Amrhein

2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Chorismate synthase catalyzes the anti-1,4-elimination of the phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate, a central building block in aromatic amino acid biosynthesis. The enzyme has an absolute requirement for reduced FMN, which in the case of the fungal chorismate synthases is supplied by an intrinsic FMN: NADPH oxidoreductase activity, i.e. these enzymes have an additional catalytic activity. Therefore, these fungal enzymes have been termed "bifunctional." We have cloned chorismate synthase from the common bread mold Neurospora crassa, expressed it heterologously in Escherichia coli, and purified it in a three-step purification procedure to homogeneity. Recombinant N. crassa chorismate synthase has a diaphorase activity, i.e. it catalyzes the reduction of oxidized FMN at the expense of NADPH. Using NADPH as a reductant, a reduced flavin intermediate was observed under single and multiple turnover conditions with spectral features similar to those reported for monofunctional chorismate synthases, thus demonstrating that the intermediate is common to the chorismate synthase-catalyzed reaction. Furthermore, multiple turnover experiments in the presence of oxygen have provided evidence that NADPH binds in or near the substrate (5-enolpyruvylshikimate 3-phosphate) binding site, suggesting that NADPH binding to bifunctional chorismate synthases is embedded in the general protein structure and a special NADPH binding domain is not required to generate the intrinsic oxidoreductase activity.Chorismate synthase catalyzes the seventh step in the shikimate pathway, which utilizes the pentose phosphate metabolite erythrose-4-phosphate and the intermediate of glycolysis, phosphoenolpyruvate, to generate aromatic amino acids and other essential aromatic compounds. The step catalyzed by chorismate synthase is formally an anti-1,4-elimination reaction of the phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate (EPSP), 1 introducing a second double bond into the ring Scheme 1 (1, 2). Although the reaction does not involve a net change in redox state, the enzyme has an absolute requirement for reduced FMN that is not consumed during substrate turnover. The role of reduced FMN has been subject to intense mechanistic studies (reviewed in Macheroux et al. (3)) that have led to a proposal of a mechanism involving radical chemistry (4, 5). Another issue revolving around the requirement for reduced flavin concerns the generation of the reduced cofactor and its sequestration by chorismate synthase. Chorismate synthases from eubacteria and plants have been shown to rely on an external source of reduced FMN, produced for example by a NAD(P)H-dependent FMN oxidoreductase. Because the reduced form of FMN binds much more tightly to the enzyme (6), it is thought that free reduced FMN is sequestered by chorismate synthase either from the cellular environment or is provided by a specific oxidoreductase. Chorismate synthases that depend on an external sou...

show abstract

Section: Methodssupporting

confidence: 86%

Section: Expression and Purification Of N Crassa Chorismate Synthasesupporting

confidence: 74%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Spectroscopic and Kinetic Characterization of the Bifunctional Chorismate Synthase from Neurospora crassa

Kitzing

Macheroux

Amrhein

2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

Enzymatic Addition/Elimination Reactions

Bugg

2012

Introduction to Enzyme and Coenzyme Chemistry

View full text Add to dashboard Cite

Radical Enzymes

Golding

2012

Encyclopedia of Radicals in Chemistry, Biology and Materials

View full text Add to dashboard Cite

Radical enzymes catalyze reactions with intermediate radicals, which are not free but bound to the enzyme. The catalytic reactions comprise generation of a radical species that initiates formation of the substrate‐derived radical, followed by conversion to the product‐related radical, and either recycling or decomposition of the initiating radical with release of the product. These characteristics are illustrated with specific radical enzymes. Ribonucleotide reductases (RNRs) use a thiyl radical to enable a common reaction mechanism for the reduction of ribose. There are three classes of RNRs that differ in the manner of thiyl radical generation: use of coenzyme B 12 , S ‐adenosylmethionine (SAM), or dioxygen with bi‐transition metal centers. Coenzyme B 12 ‐dependent eliminases and mutases are like SAM radical enzymes in that they apply the same 5′‐deoxyadenosyl radical as initiating radical, which is also used by coenzyme B 12 ‐ or SAM‐dependent RNRs. The SAM radical enzymes, which multiply in number, are involved in the maturation of enzymes and transfer of ribonucleic acids, as well as in the biosynthesis of many vitamins, coenzymes, and antibiotics. The 2‐ and 4‐hydroxyacyl‐coenzyme A (CoA) dehydratases contain a [4Fe–4S] cluster and utilize intermediate ketyl radicals, which are formed either by reduction or by combined oxidation/deprotonation of the substrates. Similar radical species participate in the reduction of benzoyl‐CoA and other aromatic compounds as well as in photolyases that repair DNA. Finally, flavin radical enzymes and two [4Fe–4S] cluster enzymes of the nonmevalonate pathway of isoprenoid biosynthesis are described. In conclusion, biotechnological applications and evolutionary aspects are discussed.

show abstract

Chorismate synthase of Neurospora crassa: A flavoprotein

Cited by 61 publications

References 20 publications

Spectroscopic and Kinetic Characterization of the Bifunctional Chorismate Synthase from Neurospora crassa

Spectroscopic and Kinetic Characterization of the Bifunctional Chorismate Synthase from Neurospora crassa

Enzymatic Addition/Elimination Reactions

Radical Enzymes

Contact Info

Product

Resources

About