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Abstract Plants release back to the atmosphere about half of the CO 2 they capture by photosynthesis. Decreasing the rate of crop respiration could therefore potentially increase yields, store more carbon in the soil and draw down atmospheric CO 2 . However, decreasing respiration rate has had very little research effort compared to increasing photosynthesis, the historically dominant metabolic paradigm for crop improvement. Conceptual and technical advances, particularly in protein turnover and directed enzyme evolution, have now opened ways to trim the large fraction of respiration that fuels proteome maintenance by lowering the breakdown and resynthesis rates of enzymes and other proteins. In addition to being theoretically possible and practicable, exploring the reduction of respiration is prudential, given that it (i) has barely yet been tried and (ii) could help meet the challenges of sustaining crop productivity and managing atmospheric carbon. 

The thiamin-requiring mutants of Arabidopsis have a storied history as a foundational model for biochemical genetics in plants and have illuminated the central role of thiamin in metabolism. Recent integrative genetic and biochemical analyses of thiamin biosynthesis and utilization imply that leaf metabolism normally operates close to thiamin-limiting conditions. Thus, the mechanisms that allocate thiamin-diphosphate (ThDP) cofactor among the diverse thiamin-dependent enzymes localized in plastids, mitochondria, peroxisomes, and the cytosol comprise an intricate thiamin economy. Here, we show that the classical thiamin-requiring 3 ( th3 ) mutant is a point mutation in plastid localized 5-deoxyxylulose synthase 1 ( DXS1 ), a key regulated enzyme in the methylerythritol 4-phosphate (MEP) isoprene biosynthesis pathway. Substitution of a lysine for a highly conserved glutamate residue (E323) located at the subunit interface of the homodimeric enzyme conditions a hypomorphic phenotype that can be rescued by supplying low concentrations of thiamin in the medium. Analysis of leaf thiamin vitamers showed that supplementing the medium with thiamin increased total ThDP content in both wild type and th3 mutant plants, supporting a hypothesis that the mutant DXS1 enzyme has a reduced affinity for the ThDP cofactor. An unexpected upregulation of a suite of biotic-stress-response genes associated with accumulation of downstream MEP intermediate MEcPP suggests that th3 causes mis-regulation of DXS1 activity in thiamin-supplemented plants. Overall, these results highlight that the central role of ThDP availability in regulation of DXS1 activity and flux through the MEP pathway. 

Abiotic stresses reduce crop growth and yield in part by disrupting metabolic homeostasis and triggering responses that change the metabolome. Experiments designed to understand the mechanisms underlying these metabolomic responses have usually not used agriculturally relevant stress regimes. We therefore subjected maize plants to drought, salt, or heat stresses that mimic field conditions and analyzed leaf responses at metabolome and transcriptome levels. Shared features of stress metabolomes included synthesis of raffinose, a compatible solute implicated in tolerance to dehydration. In addition, a marked accumulation of amino acids including proline, arginine, and γ-aminobutyrate combined with depletion of key glycolysis and tricarboxylic acid cycle intermediates indicated a shift in balance of carbon and nitrogen metabolism in stressed leaves. Involvement of the γ-aminobutyrate shunt in this process is consistent with its previously proposed role as a workaround for stress-induced thiamin-deficiency. Although convergent metabolome shifts were correlated with gene expression changes in affected pathways, patterns of differential gene regulation induced by the three stresses indicated distinct signaling mechanisms highlighting the plasticity of plant metabolic responses to abiotic stress. 

ABSTRACT Analysis of the genes retained in the minimized Mycoplasma JCVI-Syn3A genome established that systems that repair or preempt metabolite damage are essential to life. Several genes known to have such functions were identified and experimentally validated, including 5-formyltetrahydrofolate cycloligase, coenzyme A (CoA) disulfide reductase, and certain hydrolases. Furthermore, we discovered that an enigmatic YqeK hydrolase domain fused to NadD has a novel proofreading function in NAD synthesis and could double as a MutT-like sanitizing enzyme for the nucleotide pool. Finally, we combined metabolomics and cheminformatics approaches to extend the core metabolic map of JCVI-Syn3A to include promiscuous enzymatic reactions and spontaneous side reactions. This extension revealed that several key metabolite damage control systems remain to be identified in JCVI-Syn3A, such as that for methylglyoxal. IMPORTANCE Metabolite damage and repair mechanisms are being increasingly recognized. We present here compelling genetic and biochemical evidence for the universal importance of these mechanisms by demonstrating that stripping a genome down to its barest essentials leaves metabolite damage control systems in place. Furthermore, our metabolomic and cheminformatic results point to the existence of a network of metabolite damage and damage control reactions that extends far beyond the corners of it that have been characterized so far. In sum, there can be little room left to doubt that metabolite damage and the systems that counter it are mainstream metabolic processes that cannot be separated from life itself. 

Metabolic engineering uses enzymes as parts to build biosystems for specified tasks. Although a part’s working life and failure modes are key engineering performance indicators, this is not yet so in metabolic engineering because it is not known how long enzymes remain functional in vivo or whether cumulative deterioration (wear-out), sudden random failure, or other causes drive replacement. Consequently, enzymes cannot be engineered to extend life and cut the high energy costs of replacement. Guided by catalyst engineering, we adopted catalytic cycles until replacement (CCR) as a metric for enzyme functional life span in vivo. CCR is the number of catalytic cycles that an enzyme mediates in vivo before failure or replacement, i.e., metabolic flux rate/protein turnover rate. We used estimated fluxes and measured protein turnover rates to calculate CCRs for ∼100–200 enzymes each from Lactococcus lactis , yeast, and Arabidopsis . CCRs in these organisms had similar ranges (<10 3 to >10 7 ) but different median values (3–4 × 10 4 in L. lactis and yeast versus 4 × 10 5 in Arabidopsis ). In all organisms, enzymes whose substrates, products, or mechanisms can attack reactive amino acid residues had significantly lower median CCR values than other enzymes. Taken with literature on mechanism-based inactivation, the latter finding supports the proposal that 1) random active-site damage by reaction chemistry is an important cause of enzyme failure, and 2) reactive noncatalytic residues in the active-site region are likely contributors to damage susceptibility. Enzyme engineering to raise CCRs and lower replacement costs may thus be both beneficial and feasible. 

N⁶‐Formyl lysine is a well‐known modification of histones and other proteins. It can also be formed as a damaged product from direct formylation of free lysine and accompanied by other lysine derivatives such as acetylated or methylated forms. In relation to the activity of cellular repair enzymes in protein turnover and to lysine metabolism, it is important to accurately quantify the overall ratio of modified lysine to free lysine.

N⁶‐Formyl lysine was quantified using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with data collected in a non‐targeted manner using positive mode electrospray ionization on a Q‐Exactive HF⁺Orbitrap mass spectrometer. Studies were performed with lysine and deuterated lysine spiked into protein digests and solvents to investigate the extent of spontaneous formation and matrix effects of formation ofN⁶‐formyl lysine.

We show thatN⁶‐formyl lysine,N²‐formyl lysine,N⁶‐acetyl lysine, andN²‐acetyl lysine are all formed spontaneously during sample preparation and LC/MS/MS analysis, which complicates quantification of these metabolites in biological samples.N⁶‐Formyl lysine was spontaneously formed and correlated to the concentration of lysine. In the sample matrix of protein digests, 0.03% of lysine was spontaneously converted intoN⁶‐formyl lysine, and 0.005% of lysine was converted intoN⁶‐formyl lysine in pure run solvent.

Spontaneous formation ofN⁶‐formyl lysine,N⁶‐acetyl lysine,N²‐formyl lysine, andN²‐acetyl lysine needs to be subtracted from biologically formed lysine modifications when quantifying these epimetabolites in biological samples.

 

5-Deoxyribose is formed from 5′-deoxyadenosine, a toxic byproduct of radicalS-adenosylmethionine (SAM) enzymes. The degradative fate of 5-deoxyribose is unknown. Here, we define a salvage pathway for 5-deoxyribose in bacteria, consisting of phosphorylation, isomerization, and aldol cleavage steps. Analysis of bacterial genomes uncovers widespread, unassigned three-gene clusters specifying a putative kinase, isomerase, and sugar phosphate aldolase. We show that the enzymes encoded by theBacillus thuringiensiscluster, acting together in vitro, convert 5-deoxyribose successively to 5-deoxyribose 1-phosphate, 5-deoxyribulose 1-phosphate, and dihydroxyacetone phosphate plus acetaldehyde. Deleting the isomerase decreases the 5-deoxyribulose 1-phosphate pool size, and deleting either the isomerase or the aldolase increases susceptibility to 5-deoxyribose. The substrate preference of the aldolase is unique among family members, and the X-ray structure reveals an unusual manganese-dependent enzyme. This work defines a salvage pathway for 5-deoxyribose, a near-universal metabolite.

 

One way that researchers can test whether they understand a biological system is to see if they can accurately recreate it as a computer model. The more they learn about living things, the more the researchers can improve their models and the closer the models become to simulating the original. In this approach, it is best to start by trying to model a simple system. Biologists have previously succeeded in creating ‘minimal bacterial cells’. These synthetic cells contain fewer genes than almost all other living things and they are believed to be among the simplest possible forms of life that can grow on their own. The minimal cells can produce all the chemicals that they need to survive – in other words, they have a metabolism. Accurately recreating one of these cells in a computer is a key first step towards simulating a complete living system. Breuer et al. have developed a computer model to simulate the network of the biochemical reactions going on inside a minimal cell with just 493 genes. By altering the parameters of their model and comparing the results to experimental data, Breuer et al. explored the accuracy of their model. Overall, the model reproduces experimental results, but it is not yet perfect. The differences between the model and the experiments suggest new questions and tests that could advance our understanding of biology. In particular, Breuer et al. identified 30 genes that are essential for life in these cells but that currently have no known purpose. Continuing to develop and expand models like these to reproduce more complex living systems provides a tool to test current knowledge of biology. These models may become so advanced that they could predict how living things will respond to changing situations. This would allow scientists to test ideas sooner and make much faster progress in understanding life on Earth. Ultimately, these models could one day help to accelerate medical and industrial processes to save lives and enhance productivity.