Flux Analysis Uncovers Key Role of Functional Redundancy in Formaldehyde Metabolism

Abstract

Genome-scale analysis of predicted metabolic pathways has revealed the common occurrence of apparent redundancy for specific functional units, or metabolic modules. In many cases, mutation analysis does not resolve function, and instead, direct experimental analysis of metabolic flux under changing conditions is necessary. In order to use genome sequences to build models of cellular function, it is important to define function for such apparently redundant systems. Here we describe direct flux measurements to determine the role of redundancy in three modules involved in formaldehyde assimilation and dissimilation in a bacterium growing on methanol. A combination of deuterium and 14C labeling was used to measure the flux through each of the branches of metabolism for growth on methanol during transitions into and out of methylotrophy. The cells were found to differentially partition formaldehyde among the three modules depending on the flux of methanol into the cell. A dynamic mathematical model demonstrated that the kinetic constants of the enzymes involved are sufficient to account for this phenomenon. We demonstrate the role of redundancy in formaldehyde metabolism and have uncovered a new paradigm for coping with toxic, high-flux metabolic intermediates: a dynamic, interconnected metabolic loop.

QscR-Mediated Transcriptional Activation of Serine Cycle Genes in Methylobacterium extorquens AM1

“…NADP pools should then drop, releasing QscR in a noninhibited form. Flux measurements show that under these conditions, very little formaldehyde is converted directly to methylene H 4 F, with the ratio of the direct conversion route to the H 4 MPT-dependent route being 8:1 (18). Flux measurements also show that formate is produced in the H 4 MPT-linked pathway (28), and although most of it is oxidized via formate dehydrogenases, resulting in production of additional NADH (12), a small portion (12%) is converted into formyl-H 4 F, via the FtfL reaction (Fig.…”

“…4). When the switch from a multicarbon growth mode to a methylotrophic growth mode first occurs, flux measurements show that the formaldehyde produced starts flowing through the H 4 MPT-linked C 1 transfer pathway (18), resulting in production of ATP from the methanol oxidation and NAD(P)H from the methylene-H 4 MPT dehydrogenase reaction (Fig. 4A).…”

“…The linkage and co-regulation of two enzymes from this C 1 -transfer pathway with serine cycle enzymes suggested that both modules are functionally connected. This hypothesis has been supported by flux analysis data indicating that the H 4 F-linked C 1 transfer module is involved in conversion of formate to methylene-H 4 F, the starting substrate for the serine cycle (18).…”

Regulatory crosstalk of the metabolic network

“…For instance, methanotrophic bacteria use different modules to distribute toxic formaldehyde when they switch into or out of methylotrophy [13]. Indeed, environmental transitions could be the driving force for the evolution of modularity.…”

Evolution of a single gene highlights the complexity underlying molecular descriptions of fitness

“…Bridging flux to fitness provided the connections that allow the molecular attributes of a protein, like AK, to be linked to the properties of the metabolic systems under selection. 39 Resource-limited chemostat studies confirmed that growth rates ͑fitness͒ were dependent on flux through the metabolic system as determined by the respective enzyme activities. 40,41 The sensitivity of the flux ͑J͒ through a system to small changes in enzyme concentration ͑e i ͒ is described by the flux control coefficient ͑C i J ͒, A relative growth rate ͑y axis͒ can be calculated using these relations with an input of in vitro activity ͑x axis͒.…”