Heterologous production, reconstitution and EPR spectroscopic analysis of prFMN dependent enzymes

Stephen A. Marshall; Karl A.P. Payne; Karl Fisher; Deepankar Gahloth; Samuel S. Bailey; Arune Balaikaite; Annica Saaret; Irina Gostimskaya; Godwin Aleku; Huanming Huang; Stephen E.J. Rigby; David Procter; David Leys

doi:10.1016/bs.mie.2019.03.022

Heterologous production, reconstitution and EPR spectroscopic analysis of prFMN dependent enzymes

Stephen A. Marshall, Karl A.P. Payne, Karl Fisher, Deepankar Gahloth, Samuel S. Bailey, Arune Balaikaite, Annica Saaret, Irina Gostimskaya, Godwin Aleku, Huanming Huang, Stephen E.J. Rigby, David Procter, David Leys^*

^*Corresponding author for this work

The University of Manchester

UMIST

Research output: Chapter in Book/Conference proceeding › Chapter › peer-review

Abstract

The recent discovery of the prenylated FMN (prFMN) cofactor has led to a renewed interest in the prFMN-dependent UbiD family of enzymes. The latter catalyses the reversible decarboxylation of alpha-beta unsaturated carboxylic acids and features widely in microbial metabolism. The flavin prenyltransferase UbiX synthesizes prFMN from reduced FMN and phosphorylated dimethylallyl precursors. Oxidative maturation of the resulting prFMN ^reduced species to the active prFMN ^iminium form is required for UbiD activity. Heterologous production of active holo-UbiD requires co-expression of UbiX, but the levels of prFMN incorporation and oxidative maturation appear variable. Detailed protocols and strategies for in vitro reconstitution and oxidative maturation of UbiD are presented that can yield an alternative source of active holo-UbiD for biochemical studies.

Original language	English
Title of host publication	Methods in Enzymology
Pages	489-508
Number of pages	20
Volume	620
DOIs	https://doi.org/10.1016/bs.mie.2019.03.022
Publication status	Published - 2019

Publication series

Name	Methods in Enzymology
Publisher	Elsevier BV
ISSN (Print)	0076-6879

Keywords

Crystallography
Decarboxylase
EPR
Iminium
Prenyltransferase
prFMN
Radical
UbiD
UbiX

Research Beacons, Institutes and Platforms

Manchester Institute of Biotechnology

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10.1016/bs.mie.2019.03.022

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Marshall, S. A., Payne, K. A. P., Fisher, K., Gahloth, D., Bailey, S. S., Balaikaite, A., Saaret, A., Gostimskaya, I., Aleku, G., Huang, H., Rigby, S. E. J., Procter, D., & Leys, D. (2019). Heterologous production, reconstitution and EPR spectroscopic analysis of prFMN dependent enzymes. In Methods in Enzymology (Vol. 620, pp. 489-508). (Methods in Enzymology). https://doi.org/10.1016/bs.mie.2019.03.022

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title = "Heterologous production, reconstitution and EPR spectroscopic analysis of prFMN dependent enzymes",

abstract = " The recent discovery of the prenylated FMN (prFMN) cofactor has led to a renewed interest in the prFMN-dependent UbiD family of enzymes. The latter catalyses the reversible decarboxylation of alpha-beta unsaturated carboxylic acids and features widely in microbial metabolism. The flavin prenyltransferase UbiX synthesizes prFMN from reduced FMN and phosphorylated dimethylallyl precursors. Oxidative maturation of the resulting prFMN reduced species to the active prFMN iminium form is required for UbiD activity. Heterologous production of active holo-UbiD requires co-expression of UbiX, but the levels of prFMN incorporation and oxidative maturation appear variable. Detailed protocols and strategies for in vitro reconstitution and oxidative maturation of UbiD are presented that can yield an alternative source of active holo-UbiD for biochemical studies. ",

keywords = "Crystallography, Decarboxylase, EPR, Iminium, Prenyltransferase, prFMN, Radical, UbiD, UbiX",

author = "Marshall, {Stephen A.} and Payne, {Karl A.P.} and Karl Fisher and Deepankar Gahloth and Bailey, {Samuel S.} and Arune Balaikaite and Annica Saaret and Irina Gostimskaya and Godwin Aleku and Huanming Huang and Rigby, {Stephen E.J.} and David Procter and David Leys",

year = "2019",

doi = "10.1016/bs.mie.2019.03.022",

language = "English",

volume = "620",

series = "Methods in Enzymology",

publisher = "Elsevier BV",

pages = "489--508",

booktitle = "Methods in Enzymology",

}

Marshall, SA, Payne, KAP, Fisher, K, Gahloth, D, Bailey, SS, Balaikaite, A, Saaret, A, Gostimskaya, I, Aleku, G, Huang, H, Rigby, SEJ, Procter, D & Leys, D 2019, Heterologous production, reconstitution and EPR spectroscopic analysis of prFMN dependent enzymes. in Methods in Enzymology. vol. 620, Methods in Enzymology, pp. 489-508. https://doi.org/10.1016/bs.mie.2019.03.022

TY - CHAP

T1 - Heterologous production, reconstitution and EPR spectroscopic analysis of prFMN dependent enzymes

AU - Marshall, Stephen A.

AU - Payne, Karl A.P.

AU - Fisher, Karl

AU - Gahloth, Deepankar

AU - Bailey, Samuel S.

AU - Balaikaite, Arune

AU - Saaret, Annica

AU - Gostimskaya, Irina

AU - Aleku, Godwin

AU - Huang, Huanming

AU - Rigby, Stephen E.J.

AU - Procter, David

AU - Leys, David

PY - 2019

Y1 - 2019

N2 - The recent discovery of the prenylated FMN (prFMN) cofactor has led to a renewed interest in the prFMN-dependent UbiD family of enzymes. The latter catalyses the reversible decarboxylation of alpha-beta unsaturated carboxylic acids and features widely in microbial metabolism. The flavin prenyltransferase UbiX synthesizes prFMN from reduced FMN and phosphorylated dimethylallyl precursors. Oxidative maturation of the resulting prFMN reduced species to the active prFMN iminium form is required for UbiD activity. Heterologous production of active holo-UbiD requires co-expression of UbiX, but the levels of prFMN incorporation and oxidative maturation appear variable. Detailed protocols and strategies for in vitro reconstitution and oxidative maturation of UbiD are presented that can yield an alternative source of active holo-UbiD for biochemical studies.

AB - The recent discovery of the prenylated FMN (prFMN) cofactor has led to a renewed interest in the prFMN-dependent UbiD family of enzymes. The latter catalyses the reversible decarboxylation of alpha-beta unsaturated carboxylic acids and features widely in microbial metabolism. The flavin prenyltransferase UbiX synthesizes prFMN from reduced FMN and phosphorylated dimethylallyl precursors. Oxidative maturation of the resulting prFMN reduced species to the active prFMN iminium form is required for UbiD activity. Heterologous production of active holo-UbiD requires co-expression of UbiX, but the levels of prFMN incorporation and oxidative maturation appear variable. Detailed protocols and strategies for in vitro reconstitution and oxidative maturation of UbiD are presented that can yield an alternative source of active holo-UbiD for biochemical studies.

KW - Crystallography

KW - Decarboxylase

KW - EPR

KW - Iminium

KW - Prenyltransferase

KW - prFMN

KW - Radical

KW - UbiD

KW - UbiX

U2 - 10.1016/bs.mie.2019.03.022

DO - 10.1016/bs.mie.2019.03.022

M3 - Chapter

C2 - 31072499

AN - SCOPUS:85064271581

VL - 620

T3 - Methods in Enzymology

SP - 489

EP - 508

BT - Methods in Enzymology

ER -

Heterologous production, reconstitution and EPR spectroscopic analysis of prFMN dependent enzymes

Abstract

Publication series

Keywords

Research Beacons, Institutes and Platforms

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