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Article: Mechanistic insights into F

Oyugi, Mercy A / Bashiri, Ghader / Baker, Edward N / Johnson-Winters, Kayunta

Biochimica et biophysica acta. Proteins and proteomics

2017 Volume 1866, Issue 2, Page(s) 387–395

Abstract: ... ...

Abstract	F
MeSH term(s)	Bacterial Proteins/chemistry ; Citric Acid/chemistry ; Deuterium/chemistry ; Deuterium Exchange Measurement/methods ; Glucose-6-Phosphate/chemistry ; Glucosephosphate Dehydrogenase/chemistry ; Mycobacterium tuberculosis/enzymology
Chemical Substances	Bacterial Proteins ; Citric Acid (2968PHW8QP) ; Glucose-6-Phosphate (56-73-5) ; Deuterium (AR09D82C7G) ; Glucosephosphate Dehydrogenase (EC 1.1.1.49)
Language	English
Publishing date	2017-08-12
Publishing country	Netherlands
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	60-7
ISSN	1879-2596 ; 1879-260X ; 1872-8006 ; 1879-2642 ; 1879-2618 ; 1879-2650 ; 1570-9639 ; 0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 0925-4439 ; 1874-9399
ISSN (online)	1879-2596 ; 1879-260X ; 1872-8006 ; 1879-2642 ; 1879-2618 ; 1879-2650
ISSN	1570-9639 ; 0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 0925-4439 ; 1874-9399
DOI	10.1016/j.bbapap.2017.08.001
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Article ; Online: Investigating the Reaction Mechanism of F

Oyugi, Mercy A / Bashiri, Ghader / Baker, Edward N / Johnson-Winters, Kayunta

Biochemistry

2016 Volume 55, Issue 39, Page(s) 5566–5577

Abstract: ... ...

Abstract	F
MeSH term(s)	Glucosephosphate Dehydrogenase/genetics ; Glucosephosphate Dehydrogenase/metabolism ; Kinetics ; Mutagenesis, Site-Directed ; Mycobacterium tuberculosis/enzymology ; Spectrometry, Fluorescence
Chemical Substances	Glucosephosphate Dehydrogenase (EC 1.1.1.49)
Language	English
Publishing date	2016-10-04
Publishing country	United States
Document type	Journal Article
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/acs.biochem.6b00638
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Article ; Online: Optimization of Expression and Purification of Recombinant Archeoglobus fulgidus F420H2:NADP+ Oxidoreductase, an F420 Cofactor Dependent Enzyme.

Le, Cuong Quang / Joseph, Ebenezer / Nguyen, Toan / Johnson-Winters, Kayunta

The protein journal

2015 Volume 34, Issue 6, Page(s) 391–397

Abstract: Methanogens play a critical role in carbon cycling and contain a number of intriguing biosynthetic pathways. One unusual cofactor found in methanogenic and sulfate reducing archaea is Factor 420 (F420), which can be interconverted between its reduced and ...

Abstract	Methanogens play a critical role in carbon cycling and contain a number of intriguing biosynthetic pathways. One unusual cofactor found in methanogenic and sulfate reducing archaea is Factor 420 (F420), which can be interconverted between its reduced and oxidized forms by the F420H2:NADP(+) oxidoreductase (Fno) through hydride transfer mechanisms. Here, we report an optimized expression and purification method for recombinant Fno derived from the extreme thermophile Archeoglobus fulgidus. An expression vector that is codon-optimized for heterologous expression in Escherichia coli, modified growth conditions, and a modified purification protocol involving a key polyethyleneimine precipitation step results in a highly purified, homogeneous preparation of Fno that displays high catalytic activity with a truncated F420 analog. This method should accelerate studies on how Fno uses the unusual F420 cofactor during catalysis.
MeSH term(s)	Archaeal Proteins/chemistry ; Archaeal Proteins/genetics ; Archaeal Proteins/isolation & purification ; Archaeal Proteins/metabolism ; Archaeoglobus/enzymology ; Archaeoglobus/genetics ; Escherichia coli/genetics ; NADH, NADPH Oxidoreductases/chemistry ; NADH, NADPH Oxidoreductases/genetics ; NADH, NADPH Oxidoreductases/isolation & purification ; NADH, NADPH Oxidoreductases/metabolism ; NADP/metabolism ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics ; Recombinant Proteins/isolation & purification ; Recombinant Proteins/metabolism
Chemical Substances	Archaeal Proteins ; Recombinant Proteins ; NADP (53-59-8) ; NADH, NADPH Oxidoreductases (EC 1.6.-) ; F420-dependent NADP reductase (EC 1.6.8.-)
Language	English
Publishing date	2015-12
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2143071-8
ISSN	1875-8355 ; 1573-4943 ; 1572-3887
ISSN (online)	1875-8355 ; 1573-4943
ISSN	1572-3887
DOI	10.1007/s10930-015-9633-y
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Article: Effects of isoleucine 135 side chain length on the cofactor donor-acceptor distance within F

Le, Cuong Quang / Oyugi, Mercy / Joseph, Ebenezer / Nguyen, Toan / Ullah, Md Hasmat / Aubert, Joshua / Phan, Thien / Tran, Joseph / Johnson-Winters, Kayunta

Biochemistry and biophysics reports

2016 Volume 9, Page(s) 114–120

Abstract: ... ...

Abstract	F
Language	English
Publishing date	2016-11-30
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	2831046-9
ISSN	2405-5808
ISSN	2405-5808
DOI	10.1016/j.bbrep.2016.11.012
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Article: Mechanistic insights into F420-dependent glucose-6-phosphate dehydrogenase using isotope effects and substrate inhibition studies

Oyugi, Mercy A / Ghader Bashiri / Edward N. Baker / Kayunta Johnson-Winters

BBA - Proteins and Proteomics. 2017,

2017

Abstract: F420-dependent glucose-6-phosphate dehydrogenase (FGD) is involved in the committed step of the pentose phosphate pathway within mycobacteria, where it catalyzes the reaction between glucose-6-phosphate (G6P) and the F420 cofactor to yield 6- ... ...

Abstract	F420-dependent glucose-6-phosphate dehydrogenase (FGD) is involved in the committed step of the pentose phosphate pathway within mycobacteria, where it catalyzes the reaction between glucose-6-phosphate (G6P) and the F420 cofactor to yield 6-phosphogluconolactone and the reduced cofactor, F420H2. Here, we aim to probe the FGD reaction mechanism using dead-end inhibition experiments, as well as solvent and substrate deuterium isotope effects studies. The dead-end inhibition studies performed using citrate as the inhibitor revealed competitive and uncompetitive inhibition patterns for G6P and F420 respectively, thus suggesting a mechanism of ordered addition of substrates in which the F420 cofactor must first bind to FGD before G6P binding. The solvent deuterium isotope effects studies yielded normal solvent kinetic isotope effects (SKIE) on kcat and kcat/Km for both G6P and F420. The proton inventory data yielded a fractionation factor of 0.37, suggesting that the single proton responsible for the observed SKIE is likely donated by Glu109 and protonates the cofactor at position N1. The steady state substrate deuterium isotope effects studies using G6P and G6P-d1 yielded KIE of 1.1 for both kcat and kcat/Km, while the pre-steady state KIE on kobs was 1.4. Because the hydride transferred to C5 of F420 was the one targeted for isotopic substitution, these KIE values provide further evidence to support our previous findings that hydride transfer is likely not rate-limiting in the FGD reaction .
Keywords	citrates ; deuterium ; fractionation ; glucose 6-phosphate ; glucose-6-phosphate 1-dehydrogenase ; hydrides ; isotopes ; pentose phosphate cycle ; proteins ; proteomics ; protons ; solvents
Language	English
Size	p. .
Publishing place	Elsevier B.V.
Document type	Article
Note	Pre-press version
ZDB-ID	2918798-9
ISSN	1878-1454 ; 1570-9639
ISSN (online)	1878-1454
ISSN	1570-9639
DOI	10.1016/j.bbapap.2017.08.001
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Article ; Online: Elucidating the catalytic mechanism of sulfite oxidizing enzymes using structural, spectroscopic, and kinetic analyses.

Johnson-Winters, Kayunta / Tollin, Gordon / Enemark, John H

Biochemistry

2010 Volume 49, Issue 34, Page(s) 7242–7254

Abstract: Sulfite oxidizing enzymes (SOEs) are molybdenum cofactor-dependent enzymes that are found in plants, animals, and bacteria. Sulfite oxidase (SO) is found in animals and plants, while sulfite dehydrogenase (SDH) is found in bacteria. In animals, SO ... ...

Abstract	Sulfite oxidizing enzymes (SOEs) are molybdenum cofactor-dependent enzymes that are found in plants, animals, and bacteria. Sulfite oxidase (SO) is found in animals and plants, while sulfite dehydrogenase (SDH) is found in bacteria. In animals, SO catalyzes the oxidation of toxic sulfite to sulfate as the final step in the catabolism of the sulfur-containing amino acids, methionine and cysteine. In humans, sulfite oxidase deficiency is an inherited recessive disorder that produces severe neonatal neurological problems that lead to early death. Plant SO (PSO) also plays an important role in sulfite detoxification and in addition serves as an intermediate enzyme in the assimilatory reduction of sulfate. In vertebrates, the proposed catalytic mechanism of SO involves two intramolecular one-electron transfer (IET) steps from the molybdenum cofactor to the iron of the integral b-type heme. A similar mechanism is proposed for SDH, involving its molybdenum cofactor and c-type heme. However, PSO, which lacks an integral heme cofactor, uses molecular oxygen as its electron acceptor. Here we review recent results for SOEs from kinetic measurements, computational studies, electron paramagnetic resonance (EPR) spectroscopy, electrochemical measurements, and site-directed mutagenesis on active site residues of SOEs and of the flexible polypepetide tether that connects the heme and molybdenum domains of human SO. Rapid kinetic studies of PSO are also discussed.
MeSH term(s)	Animals ; Binding Sites ; Catalysis ; Coenzymes ; Electron Spin Resonance Spectroscopy ; Electron Transport ; Heme/analogs & derivatives ; Heme/chemistry ; Heme/metabolism ; Humans ; Kinetics ; Metalloproteins ; Molybdenum/chemistry ; Mutagenesis, Site-Directed ; Oxidation-Reduction ; Oxidoreductases Acting on Sulfur Group Donors ; Pteridines ; Spectrum Analysis ; Sulfite Dehydrogenase/chemistry ; Sulfite Dehydrogenase/metabolism ; Sulfite Oxidase/chemistry ; Sulfite Oxidase/metabolism ; Sulfites/metabolism
Chemical Substances	Coenzymes ; Metalloproteins ; Pteridines ; Sulfites ; heme C (26598-29-8) ; Heme (42VZT0U6YR) ; Molybdenum (81AH48963U) ; molybdenum cofactor (ATN6EG42UQ) ; Oxidoreductases Acting on Sulfur Group Donors (EC 1.8.-) ; Sulfite Dehydrogenase (EC 1.8.2.1) ; SUOX protein, human (EC 1.8.3.1) ; Sulfite Oxidase (EC 1.8.3.1)
Language	English
Publishing date	2010-07-27
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Review
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/bi1008485
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Article: Investigating the Reaction Mechanism of F420-Dependent Glucose-6-phosphate Dehydrogenase from Mycobacterium tuberculosis: Kinetic Analysis of the Wild-Type and Mutant Enzymes

Oyugi, Mercy A / Bashiri Ghader / Baker Edward N / Johnson-Winters Kayunta

Biochemistry. 2016 Oct. 04, v. 55, no. 39

2016

Abstract: Fâââ-dependent glucose-6-phosphate dehydrogenase (FGD) catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, using Fâââ cofactor as the hydride transfer acceptor, within mycobacteria. A previous crystal structure ... ...

Abstract	Fâââ-dependent glucose-6-phosphate dehydrogenase (FGD) catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, using Fâââ cofactor as the hydride transfer acceptor, within mycobacteria. A previous crystal structure of wild-type FGD led to a proposed mechanism suggesting that the active site residues His40, Trp44, and Glu109 could be involved in catalysis. We have characterized the wild-type FGD and five FGD variants (H40A, W44F, W44Y, W44A, and E109Q) by fluorescence binding assays and steady-state and pre-steady-state kinetic experiments. Compared to wild-type FGD, all the variants had lower binding affinities for Fâââ, thus suggesting that Trp44, His40, and Glu109 aid in Fâââ binding. While all the variants had decreased catalytic efficiencies, FGD H40A and W44A were the least efficient, having lost â¼1000- and â¼2000-fold activity, respectively. This confirms a crucial catalytic role for His40 in the FGD reaction and suggests that aromaticity at residue 44 aids catalysis. To investigate the proposed roles of Glu109 and His40 in acidâbase catalysis, the pH dependence of kinetic parameters has been determined for the E109Q and H40A mutants and compared to those of the wild-type enzyme. The log kcâââpH profile of wild-type FGD and E109Q revealed two ionizable residues in the enzymeâsubstrate complex, while H40A displayed only one ionization event. The FGD E109Q variant displayed pH-dependent kinetic cooperativity with respect to the Fâââ cofactor. The multiple-turnover pre-steady-state kinetics were biphasic for wild-type FGD, W44F, W44Y, and E109Q, while the H40A and W44A variants displayed only a single phase because of their reduced catalytic efficiency.
Keywords	Mycobacterium tuberculosis ; active sites ; binding capacity ; catalytic activity ; crystal structure ; enzyme substrates ; fluorescence ; glucose 6-phosphate ; glucose-6-phosphate 1-dehydrogenase ; hydrides ; ionization ; kinetics ; mutants ; pH
Language	English
Dates of publication	2016-1004
Size	p. 5566-5577.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021%2Facs.biochem.6b00638
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Article ; Online: Kinetic results for mutations of conserved residues H304 and R309 of human sulfite oxidase point to mechanistic complexities.

Davis, Amanda C / Johnson-Winters, Kayunta / Arnold, Anna R / Tollin, Gordon / Enemark, John H

Metallomics : integrated biometal science

2014 Volume 6, Issue 9, Page(s) 1664–1670

Abstract: Several point mutations in the gene of human sulfite oxidase (hSO) result in isolated sulfite oxidase deficiency, an inherited metabolic disorder. Three conserved residues (H304, R309, K322) are hydrogen bonded to the phosphate group of the molybdenum ... ...

Abstract	Several point mutations in the gene of human sulfite oxidase (hSO) result in isolated sulfite oxidase deficiency, an inherited metabolic disorder. Three conserved residues (H304, R309, K322) are hydrogen bonded to the phosphate group of the molybdenum cofactor, and the R309H and K322R mutations are responsible for isolated sulfite oxidase deficiency. The kinetic effects of the K322R mutation have been previously reported (Rajapakshe et al., Chem. Biodiversity, 2012, 9, 1621-1634); here we investigate several mutants of H304 and R309 by steady-state kinetics, laser flash photolysis studies of intramolecular electron transfer (IET), and spectroelectrochemistry. An unexpected result is that all of the mutants show decreased rates of IET but increased steady-state rates of catalysis. However, in all cases the rate of IET is greater than the overall turnover rate, showing that IET is not the rate determining step for any of the mutations.
MeSH term(s)	Arginine/genetics ; Conserved Sequence ; Crystallography, X-Ray ; Electrochemistry ; Electrons ; Histidine/genetics ; Humans ; Iron/metabolism ; Kinetics ; Models, Molecular ; Mutant Proteins/metabolism ; Mutation/genetics ; Oxidation-Reduction ; Oxidoreductases Acting on Sulfur Group Donors/genetics ; Spectrum Analysis
Chemical Substances	Mutant Proteins ; Histidine (4QD397987E) ; Arginine (94ZLA3W45F) ; Iron (E1UOL152H7) ; Oxidoreductases Acting on Sulfur Group Donors (EC 1.8.-) ; SUOX protein, human (EC 1.8.3.1)
Language	English
Publishing date	2014-06-26
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2474317-3
ISSN	1756-591X ; 1756-5901
ISSN (online)	1756-591X
ISSN	1756-5901
DOI	10.1039/c4mt00099d
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Article ; Online: Evidence of Negative Cooperativity and Half-Site Reactivity within an F420-Dependent Enzyme: Kinetic Analysis of F420H2:NADP(+) Oxidoreductase.

Joseph, Ebenezer / Le, Cuong Quang / Nguyen, Toan / Oyugi, Mercy / Hossain, Mohammad Shawkat / Foss, Frank W / Johnson-Winters, Kayunta

Biochemistry

2016 Volume 55, Issue 7, Page(s) 1082–1090

Abstract: Here, we report the very first example of half-site reactivity and negative cooperativity involving an important F420 cofactor-dependent enzyme. F420H2:NADP(+) oxidoreductase (Fno) is an F420 cofactor-dependent enzyme that catalyzes the reversible ... ...

Abstract	Here, we report the very first example of half-site reactivity and negative cooperativity involving an important F420 cofactor-dependent enzyme. F420H2:NADP(+) oxidoreductase (Fno) is an F420 cofactor-dependent enzyme that catalyzes the reversible reduction of NADP(+) through the transfer of a hydride from the reduced F420 cofactor. These catalytic processes are of major significance in numerous biochemical processes. While the steady-state kinetic analysis showed classic Michaelis-Menten kinetics with varying concentrations of the F420 redox moiety, FO, such plots revealed non-Michaelis-Menten kinetic behavior when NADPH was varied. The double reciprocal plot of the varying concentrations of NADPH displays a downward concave shape, suggesting that negative cooperativity occurs between the two identical monomers. The transient state kinetic data show a burst prior to entering steady-state turnover. The burst suggests that product release is rate-limiting, and the amplitude of the burst phase corresponds to production of product in only one of the active sites of the functional dimer. These results suggest either half-site reactivity or an alternate sites model wherein the reduction of the cofactor, FO occurs at one active site at a time followed by reduction at the second active site. Thus, the data imply that Fno may be a functional regulatory enzyme.
MeSH term(s)	Algorithms ; Archaeal Proteins/chemistry ; Archaeal Proteins/genetics ; Archaeal Proteins/metabolism ; Archaeoglobus fulgidus/enzymology ; Biocatalysis ; Catalytic Domain ; Dimerization ; Hydrogen Bonding ; Ligands ; Models, Molecular ; NADH, NADPH Oxidoreductases/chemistry ; NADH, NADPH Oxidoreductases/genetics ; NADH, NADPH Oxidoreductases/metabolism ; NADP/metabolism ; Oxidation-Reduction ; Protein Conformation ; Recombinant Proteins/chemistry ; Recombinant Proteins/metabolism ; Riboflavin/analogs & derivatives ; Riboflavin/metabolism ; Spectrometry, Fluorescence
Chemical Substances	Archaeal Proteins ; Ligands ; Recombinant Proteins ; NADP (53-59-8) ; coenzyme F420 (64885-97-8) ; NADH, NADPH Oxidoreductases (EC 1.6.-) ; F420-dependent NADP reductase (EC 1.6.8.-) ; Riboflavin (TLM2976OFR)
Language	English
Publishing date	2016-02-23
Publishing country	United States
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/acs.biochem.5b00762
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Article ; Online: Convenient synthesis of deazaflavin cofactor FO and its activity in F(420)-dependent NADP reductase.

Hossain, Mohammad S / Le, Cuong Q / Joseph, Ebenezer / Nguyen, Toan Q / Johnson-Winters, Kayunta / Foss, Frank W

Organic & biomolecular chemistry

2015 Volume 13, Issue 18, Page(s) 5082–5085

Abstract: F420 and FO are phenolic 5-deazaflavin cofactors that complement nicotinamide and flavin redox coenzymes in biochemical oxidoreductases and photocatalytic systems. Specifically, these 5-deazaflavins lack the single electron reactivity with O2 of ... ...

Abstract	F420 and FO are phenolic 5-deazaflavin cofactors that complement nicotinamide and flavin redox coenzymes in biochemical oxidoreductases and photocatalytic systems. Specifically, these 5-deazaflavins lack the single electron reactivity with O2 of riboflavin-derived coenzymes (FMN and FAD), and, in general, have a more negative redox potential than NAD(P)(+). For example, F420-dependent NADP(+) oxidoreductase (Fno) is critical to the conversion of CO2 to CH4 by methanogenic archaea, while FO functions as a light-harvesting agent in DNA repair. The preparation of these cofactors is an obstacle to their use in biochemical studies and biotechnology. Here, a convenient synthesis of FO was achieved by improving the redox stability of synthetic intermediates containing a polar, electron-rich aminophenol fragment. Improved yields and simplified purification techniques for FO are described. Additionally, Fno activity was restored with FO in the absence of F420. Investigating the FO-dependent NADP(+)/NADPH redox process by stopped-flow spectrophotometry, steady state kinetics were defined as having a Km of 4.00 ± 0.39 μM and a kcat of 5.27 ± 0.14 s(-1). The preparation of FO should enable future biochemical studies and novel uses of F420 mimics.
MeSH term(s)	NADP/chemistry ; Oxidoreductases/chemistry ; Riboflavin/analogs & derivatives ; Riboflavin/chemistry
Chemical Substances	factor 420 (37333-48-5) ; NADP (53-59-8) ; Oxidoreductases (EC 1.-) ; Riboflavin (TLM2976OFR)
Language	English
Publishing date	2015-05-14
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2097583-1
ISSN	1477-0539 ; 1477-0520
ISSN (online)	1477-0539
ISSN	1477-0520
DOI	10.1039/c5ob00365b
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