Toward an evaluation of metabolite channeling in vivo

https://doi.org/10.1016/j.copbio.2019.09.013Get rights and content

Highlights

  • Intermediate metabolites are channeled between metabolic pathway enzymes.

  • Metabolite channeling is a promising tool to regulate metabolic pathway fluxes.

  • Metabolic flux analyses can capture channeling signatures in a metabolic network.

  • Evaluation of channeling effects in vivo requires multiplex approaches.

Intermediates of metabolic pathways are sometimes contained within cavities of enzyme molecules and passed directly from active centers of one to next enzymes without diffusing into bulk matrix. This ‘metabolite channeling’ is postulated to have various advantages in enhancing and regulating pathway reactions and considered as a central unit to control metabolic network. Therefore, it has a strong potential in applications to metabolic engineering to enhance the production of desired molecules. Quantitative evaluation of the effects of metabolite channeling is crucial for its appropriate application and further understanding of its functions. In the present review, current approaches to demonstrate functional metabolite channeling will be reviewed and their extension toward quantitative evaluation of channeling effects in vivo will be discussed.

Introduction

Metabolic enzymes catalyzing sequential reactions of a metabolic pathway often form multi-enzyme complex, sometimes called a ‘metabolon’ [1], in which intermediate metabolites are directly passed from catalytic cores of the first to the second enzyme without diffusing into the bulk matrix. This inclusion of intermediate metabolites within a multi-enzyme complex or a multi-functional protein is metabolite channeling, also called substrate channeling or metabolic channeling. Various functions of metabolite channeling have been proposed (Figure 1). First, metabolite channeling increases intermediate concentration near the reaction centers of the second enzyme. This functions to enhance pathway reactions by 1) increasing reaction rates; 2) shortening transition time; and 3) overcoming thermodynamically infeasible equilibrium within a physiological metabolite concentration range (Figure 1a). Second, multi-enzyme complexes physically contain intermediates within the molecules and prevent the access by external molecules. This functions in 4) protecting unstable intermediates from degradation; 5) preventing cytotoxic reactions between intermediates and cellular components; 6) protecting reaction centers from inhibitors; 7) determining metabolic flux distribution through competing pathways by restricting the access of enzymes to the intermediates; and 8) limiting ionization of intermediates by excluding water molecules from the reaction (Figure 1b). The function in 9) shortening diffusion distance for intermediates to accelerate reactions (Figure 1c) is considered mostly unlikely due to much shorter time required for diffusion of small molecules than that for enzyme reactions [2,3]. Nevertheless, it may function in specific microenvironments, including highly condensed, limited water availability, or highly compartmented environments, which can significantly limit the diffusion of intermediates [4].

Metabolite channeling is considered as a central control mechanism of cellular metabolism. It also draws attention as a promising tool to enhance and regulate productions of desired molecules in chemical and biological engineering [5,6]. Especially in the last few years, various techniques have been developed to artificially organize multi-enzyme complexes in vitro and in vivo (reviewed in a recent book [7••]) and successfully applied to gain higher production of desired molecules [5,8]. Quantitative evaluation of the effects of metabolite channeling in metabolic flux modulation is desired to understand its functions and potentials. Whereas this is a technically very difficult task, recent advances in structural biology, computational modeling, and metabolic flux analysis bear potential for innovation in metabolite channeling studies. In this review, current approaches to demonstrate metabolite channeling are reviewed with the examples of recent studies. The strategies toward the quantitative understanding of metabolic and physiological functions of metabolite channeling will also be discussed.

Section snippets

Experimental approaches to demonstrate metabolite channeling

The experimental approaches to examine the occurrence of metabolite channeling are described as 1) enzyme kinetics analysis, 2) isotope dilution/ enrichment analysis, 3) enzyme competition assay, 4) inhibitor resistance assay, and 5) quantification of intermediate in the bulk phase (Figure 2) [9, 10, 11]. Since all the methodologies are indirect [9] and test only one function of channeling, combination of more than one proof is crucial. In addition, protein structure analyses often support

Capturing metabolite channeling using metabolic flux analysis

Metabolic flux analysis (MFA) analyzes isotope label (typically 13C) redistribution in metabolic network following feeding of isotopically labeled substrates. Since label redistribution is sensitive to channeling [26], MFA can be a tool to demonstrate metabolite channeling. Steady-state MFA is an approach to fit estimated metabolic fluxes in a metabolic network model to experimentally determined 13C re-distribution pattern of metabolites at an isotopic steady state. The effects of the

Metabolite channeling in flux balance analysis

Flux balance analysis (FBA) is another method to estimate metabolic flux distribution in a metabolic model in the constrained flux space defined by mass conservation and pre-specified reversibility of the reactions [33]. Thermodynamics of metabolic reactions is often used to add an additional layer of constraints in FBA [34]. However, channeling can overcome thermodynamic penalty and cause inconsistency between measured and estimated fluxes. For example, malate oxidation catalyzed by MDH in the

Toward a quantitative evaluation of metabolite channeling in vivo

One of the overarching goals of metabolite channeling study is an accurate evaluation of metabolic and physiological effects of metabolite channeling in vivo. Since measurement of metabolite contents and enzyme in subcellular compartments is technically difficult, application of in vitro kinetics results to living systems can have critical pitfalls. Assuming the transient nature of multi-enzyme complex mediating metabolite channeling [40], quantitative assessment of channeling effects is

Conclusions

Despite of remaining difficulties, combination of current analytical technologies will make the evaluation of metabolite channeling effects in metabolic network feasible. This will lead to more complete understanding of the mechanism of metabolic regulation, more precise evaluation of metabolic flux changes, and additional tools to design metabolic network for metabolic engineering. These will enable rational design of metabolite channeling and enhance its application in biotechnology.

Conflict of interest statement

Nothing declared.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

This work was supported by the National Science Foundation [grant number 1845451] and University of Nebraska-Lincoln Faculty Startup Grant.

References (44)

  • J.E. Bassard et al.

    How to prove the existence of metabolons?

    Phytochem Rev

    (2018)
  • G. Ke et al.

    Directional regulation of enzyme pathways through the control of substrate channeling on a DNA origami scaffold

    Angew Chemie Int Ed

    (2016)
  • L. Poshyvailo et al.

    Does metabolite channeling accelerate enzyme-catalyzed cascade reactions?

    PLoS One

    (2017)
  • L.J. Sweetlove et al.

    The spatial organization of metabolism within the plant cell

    Annu Rev Plant Biol

    (2013)
  • M.J. Lee et al.

    Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm

    Nat Chem Biol

    (2018)
  • N. Smirnoff

    Engineering of metabolic pathways using synthetic enzyme complexes

    Plant Physiol

    (2019)
  • J.L. Lin et al.

    Synthetic protein scaffolds for biosynthetic pathway colocalization on lipid droplet membranes

    ACS Synth Biol

    (2017)
  • I. Wheeldon et al.

    Substrate channelling as an approach to cascade reactions

    Nat Chem

    (2016)
  • L.J. Sweetlove et al.

    The role of dynamic enzyme assemblies and substrate channelling in metabolic regulation

    Nat Commun

    (2018)
  • H.O. Spivey et al.

    Substrate channeling

    Methods

    (1999)
  • J.J. Tanner

    Structural biology of proline catabolic enzymes

    Antioxid Redox Signal

    (2019)
  • Cited by (16)

    • Mimicking natural strategies to create multi-environment enzymatic reactors: From natural cell compartments to artificial polyelectrolyte reactors

      2022, Biotechnology Advances
      Citation Excerpt :

      Indeed, in multi-enzyme complexes, substrate channeling attains high local intermediate substrate concentrations near the reaction centers of the appropriate enzyme. This mechanism increases reaction rates and shortens transition time, regulates competition between different pathways for common metabolites, protects unstable intermediates from degradation, and inhibits toxic reactions between intermediates and cellular components (Obata, 2020; Raushel et al., 2003; Wheeldon et al., 2016). The distance between enzymes has a significant effect on the rate of reactions via substrate channeling in sequential enzymatic reactions (Hwang and Lee, 2019).

    • Engineering microbial metabolic energy homeostasis for improved bioproduction

      2021, Biotechnology Advances
      Citation Excerpt :

      This strategy enabled increased production of isopentenol in E. coli (Kang et al., 2016) (Fig. 4A). Other studies have reported that metabolite channeling can be used to engineer ME-saving pathways by reducing the burden of enzyme expression, shortening the diffusion distance for intermediates, and mitigating toxic metabolite inhibition (Obata, 2020; Zhang, 2011). For example, metabolite channeling of three mevalonate biosynthetic enzymes was achieved in E. coli through the formation of a synthetic complex, which reduced bioenergetic load with low enzyme expression, resulting in a 77-fold increase in the mevalonate titer (Dueber et al., 2009).

    • Regulation | Metabolite channeling in energy metabolism

      2021, Encyclopedia of Biological Chemistry: Third Edition
    • Microenvironmental engineering: An effective strategy for tailoring enzymatic activities

      2020, Chinese Journal of Chemical Engineering
      Citation Excerpt :

      More detailed discussions on proximity channeling can be found in a series of recent reviews by Zhang and Hess [73], Rabe et al. [74], Sweetlove and Fernie [75], and research reports by Eun et al. [76], Kuzmak et al. [77], etc. It is worth noting that the steady state of a cascade reaction does not depend on channeling [78–80], thus a steady-state enhancement of the overall production requires an increased activity of the rate-limiting enzymes. The observed steady-state enhancement thus implies that scaffolds have an effect on the activity of immobilized enzymes rather than the intermediate transport.

    • Dynamics in redox metabolism, from stoichiometry towards kinetics

      2020, Current Opinion in Biotechnology
      Citation Excerpt :

      Unfortunately, limited quantitative data are available for compartment specific concentrations. In addition, metabolite channelling may overcome thermodynamic constraints by creating local high concentration environments near enzyme reaction centres, enabling thermodynamic pathway feasibility which may not be observed from the measured overall cellular metabolite concentrations [9,10]. Here, we will review recent studies providing insights into the dynamics of the redox metabolism upon changes in the environment, discuss the relevance of the understanding of the redox metabolism for the design and optimization of microbial production strains and processes, and describe how technical challenges in measuring redox states in different cellular compartments can be approached.

    View all citing articles on Scopus
    View full text