How to prove the existence of metabolons?

Abstract

Sequential enzymes in biosynthetic pathways are organized in metabolons. It is challenging to provide experimental evidence for the existence of metabolons as biosynthetic pathways are composed of highly dynamic protein–protein interactions. Many different methods are being applied, each with strengths and weaknesses. We will present and evaluate several techniques that have been applied in providing evidence for the orchestration of the biosynthetic pathways of cyanogenic glucosides and glucosinolates in metabolons. These evolutionarily related pathways have ER-localized cytochromes P450 that are proposed to function as anchoring site for assembly of the enzymes into metabolons. Additionally, we have included commonly used techniques, even though they have not been used (yet) on these two pathways. In the review, special attention will be given to less-exploited fluorescence-based methods such as FCS and FLIM. Ultimately, understanding the orchestration of biosynthetic pathways may contribute to successful engineering in heterologous hosts.

Characterization of Arabidopsis CYP79C1 and CYP79C2 by Glucosinolate Pathway Engineering in Nicotiana benthamiana Shows Substrate Specificity Toward a Range of Aliphatic and Aromatic Amino Acids

“…However, when the aliphatic core pathway was supplemented with the chain elongation pathway, neither of the two CYP79Cs used phenylalanine as precursor at all. Other differences were also observed: CYP79C2 showed substrate specificity to homo-and dihomoleucine, while CYP79C1 stopped metabolizing nonchain-elongated amino acids and apparently exclusively channeled dihomoleucine and dihomomethionine into GLS biosynthesis (Figures 2-4, Bassard and Halkier, 2018). A possible explanation for why the CYP79C specificities depended so strongly on the co-expressed biosynthetic pathways could be competition for available substrates.…”

“…P-values (two-sided Student's t-test) used to compare the same type of GLS yields between B and C are listed in Supplementary Table S8. subcellular localization (Sønderby et al, 2010), the variation may reflect the ability to obtain physical proximity between the CYP79s and the remaining core pathway enzymes in the heterologous tobacco host (Bassard and Halkier, 2018).…”

The Phenylpropanoid Case – It Is Transport That Matters

“…The cooperating enzymes from the phenylpropanoid pathway were proposed to be organized into complexes called metabolons, and a number of reviews may serve as evidence in this respect ( Winkel-Shirley, 1999 ; Jørgensen et al, 2005 ; Sweetlove and Fernie, 2013 ; Laursen et al, 2015 ; Bassard and Halkier, 2018 ). The term “metabolon” encompasses multienzymatic complexes bound to the cellular structural elements – membranes.…”

Unravelling Protein-Protein Interaction Networks Linked to Aliphatic and Indole Glucosinolate Biosynthetic Pathways in Arabidopsis

“…Within metabolism, many biosynthetic pathways are proposed to be assembled into multi-enzyme complexes called metabolons (Srere, 1985 ). Organization into metabolons provides several advantages such as increase in local concentrations of the enzymes and their substrates, efficient sequestration of reactive intermediates and improved channeling of intermediates to increase flux (Bassard et al, 2012 ; Laursen et al, 2015 , 2016 ; Dastmalchi et al, 2016 ; French et al, 2016 ), and there is increasing evidence for the existence of pathway-specific metabolons (Bassard and Halkier, 2017 ). Within the crowded intracellular environment where proteins are constantly coming into physical contact, it is an open question how multi-enzyme pathways are orchestrated in response to developmental and exogenous cues.…”