Kinetic characterization of an organic radical in the ascarylose biosynthetic pathway

David A. Johnson, George T. Gassner, Vahe Bandarian, Frank J. Ruzicka, David P. Ballou, George H. Reed, Hung Wen Liu

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

The lipopolysaccharide of Yersinia pseudotuberculosis V includes a 3, 6- dideoxyhexose, ascarylose, as the nonreducing end of the O-antigen tetrasaccharide. The C-3 deoxygenation of CDP-6-deoxy-L-threo-D-glycero-4- hexulose is a critical reaction in the biosynthesis of ascarylose. The first half of the reaction is a dehydration catalyzed by CDP-6-deoxy-L-threo-D- glycero-4-hexulose-3-dehydrase (E1), which is PMP-dependent and contains a redox-active [2Fe-2S] center. The second half is a reduction that requires an additional enzyme, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3, formerly known as CDP-6-deoxy-Δ3,4-glucoseen reductase), which has a FAD and a [2Fe-2S] center in the active site. Using NADH as the reductant in the coupled E1-E3 reaction, we have monitored the kinetics of a radical intermediate using both stopped-flow spectrophotometry and rapid freeze quench EPR under aerobic and hypoxic conditions. In the EPR studies, a sharp signal at g = 2.003 was found to appear at a rate which is kinetically competent, reaching its maximum intensity at ~150 ms. Stopped-flow UV-vis analysis of the reaction elucidated a minimum of six optically distinguishable states in the mechanism of electron transfer from NADH to substrate. Interestingly, one of the detected intermediates has a time course nearly identical to that of the radical detected by rapid freeze-quench EPR. The difference UV-vis spectrum of this intermediate displays a maximum at 456 nm with a shoulder at 425 nm. Overall, these results are consistent with an electron transfer pathway that includes a radical intermediate with the unpaired spin localized on the substrate-cofactor complex. Evidence in support of this mechanism is presented in this report. These studies add the PMP-glucoseen radical to the growing list of mechanistically important bioorganic radical intermediates that have recently been discovered.

Original languageEnglish (US)
Pages (from-to)15846-15856
Number of pages11
JournalBiochemistry
Volume35
Issue number49
DOIs
StatePublished - 1996
Externally publishedYes

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Biosynthetic Pathways
Cytidine Diphosphate
Paramagnetic resonance
NAD
Kinetics
Electrons
Yersinia pseudotuberculosis
O Antigens
Flavin-Adenine Dinucleotide
Spectrophotometry
Biosynthesis
Reducing Agents
Substrates
Dehydration
Oxidation-Reduction
Lipopolysaccharides
Catalytic Domain
Oxidoreductases
Display devices
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Johnson, D. A., Gassner, G. T., Bandarian, V., Ruzicka, F. J., Ballou, D. P., Reed, G. H., & Liu, H. W. (1996). Kinetic characterization of an organic radical in the ascarylose biosynthetic pathway. Biochemistry, 35(49), 15846-15856. https://doi.org/10.1021/bi961370w

Kinetic characterization of an organic radical in the ascarylose biosynthetic pathway. / Johnson, David A.; Gassner, George T.; Bandarian, Vahe; Ruzicka, Frank J.; Ballou, David P.; Reed, George H.; Liu, Hung Wen.

In: Biochemistry, Vol. 35, No. 49, 1996, p. 15846-15856.

Research output: Contribution to journalArticle

Johnson, DA, Gassner, GT, Bandarian, V, Ruzicka, FJ, Ballou, DP, Reed, GH & Liu, HW 1996, 'Kinetic characterization of an organic radical in the ascarylose biosynthetic pathway', Biochemistry, vol. 35, no. 49, pp. 15846-15856. https://doi.org/10.1021/bi961370w
Johnson, David A. ; Gassner, George T. ; Bandarian, Vahe ; Ruzicka, Frank J. ; Ballou, David P. ; Reed, George H. ; Liu, Hung Wen. / Kinetic characterization of an organic radical in the ascarylose biosynthetic pathway. In: Biochemistry. 1996 ; Vol. 35, No. 49. pp. 15846-15856.
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AB - The lipopolysaccharide of Yersinia pseudotuberculosis V includes a 3, 6- dideoxyhexose, ascarylose, as the nonreducing end of the O-antigen tetrasaccharide. The C-3 deoxygenation of CDP-6-deoxy-L-threo-D-glycero-4- hexulose is a critical reaction in the biosynthesis of ascarylose. The first half of the reaction is a dehydration catalyzed by CDP-6-deoxy-L-threo-D- glycero-4-hexulose-3-dehydrase (E1), which is PMP-dependent and contains a redox-active [2Fe-2S] center. The second half is a reduction that requires an additional enzyme, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3, formerly known as CDP-6-deoxy-Δ3,4-glucoseen reductase), which has a FAD and a [2Fe-2S] center in the active site. Using NADH as the reductant in the coupled E1-E3 reaction, we have monitored the kinetics of a radical intermediate using both stopped-flow spectrophotometry and rapid freeze quench EPR under aerobic and hypoxic conditions. In the EPR studies, a sharp signal at g = 2.003 was found to appear at a rate which is kinetically competent, reaching its maximum intensity at ~150 ms. Stopped-flow UV-vis analysis of the reaction elucidated a minimum of six optically distinguishable states in the mechanism of electron transfer from NADH to substrate. Interestingly, one of the detected intermediates has a time course nearly identical to that of the radical detected by rapid freeze-quench EPR. The difference UV-vis spectrum of this intermediate displays a maximum at 456 nm with a shoulder at 425 nm. Overall, these results are consistent with an electron transfer pathway that includes a radical intermediate with the unpaired spin localized on the substrate-cofactor complex. Evidence in support of this mechanism is presented in this report. These studies add the PMP-glucoseen radical to the growing list of mechanistically important bioorganic radical intermediates that have recently been discovered.

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