The number of catalytic cycles in an enzyme's lifetime and why it matters to metabolic engineering.

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 (<103 to >107) but different median values (3-4 × 104 in L. lactis and yeast versus 4 × 105 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.

In vivo homopropargylglycine incorporation enables sampling, isolation and characterization of nascent proteins from Arabidopsis thaliana.

Determining which proteins are actively synthesized at a given point in time and extracting a representative sample for analysis is important to understand plant responses. Here we show that the methionine (Met) analogue homopropargylglycine (HPG) enables Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) of a small sample of the proteins being synthesized in Arabidopsis plants or cell cultures, facilitating their click-chemistry enrichment for analysis. The sites of HPG incorporation could be confirmed by peptide mass spectrometry at Met sites throughout protein amino acid sequences and correlation with independent studies of protein labelling with 15 N verified the data. We provide evidence that HPG-based BONCAT tags a better sample of nascent plant proteins than azidohomoalanine (AHA)-based BONCAT in Arabidopsis and show that the AHA induction of Met metabolism and greater inhibition of cell growth rate than HPG probably limits AHA incorporation at Met sites in Arabidopsis. We show HPG-based BONCAT provides a verifiable method for sampling, which plant proteins are being synthesized at a given time point and enriches a small portion of new protein molecules from the bulk protein pool for identification, quantitation and subsequent biochemical analysis. Enriched nascent polypeptides samples were found to contain significantly fewer common post-translationally modified residues than the same proteins from whole plant extracts, providing evidence for age-related accumulation of post-translational modifications in plants.

How is auxin linked with cellular energy pathways to promote growth?

Auxin is the 'growth hormone' and modulation of its concentration correlates with changes in photosynthesis and respiration, influencing the cellular energy budget for biosynthesis and proliferation. However, the relative importance of mechanisms by which auxin directly influences photosynthesis and respiration, or vice versa, are unclear. Here we bring together recent evidence linking auxin with photosynthesis, plastid biogenesis, mitochondrial metabolism and retrograde signalling and through it we propose three hypotheses to test to unify current findings. These require delving into the control of auxin conjugation to primary metabolic intermediates, translational control under auxin regulation and post-translational influences of auxin on primary metabolic processes.

Knockdown of Succinate Dehydrogenase Assembly Factor 2 Induces Reactive Oxygen Species-Mediated Auxin Hypersensitivity Causing pH-Dependent Root Elongation.

Metabolism, auxin signaling and reactive oxygen species (ROS) all contribute to plant growth, and each is linked to plant mitochondria and the process of respiration. Knockdown of mitochondrial succinate dehydrogenase assembly factor 2 (SDHAF2) in Arabidopsis thaliana lowered succinate dehydrogenase activity and led to pH-inducible root inhibition when the growth medium pH was poised at different points between 7.0 and 5.0, but this phenomenon was not observed in wildtype (WT). Roots of sdhaf2 mutants showed high accumulation of succinate, depletion of citrate and malate and up-regulation of ROS-related and stress-inducible genes at pH 5.5. A change of oxidative status in sdhaf2 roots at low pH was also evidenced by low ROS staining in root tips and altered root sensitivity to H2O2. sdhaf2 had low auxin activity in root tips via DR5-GUS staining but displayed increased indole-3-acetic acid (IAA, auxin) abundance and IAA hypersensitivity, which is most likely caused by the change in ROS levels. On this basis, we conclude that knockdown of SDHAF2 induces pH-related root elongation and auxin hyperaccumulation and hypersensitivity, mediated by altered ROS homeostasis. This observation extends the existing evidence of associations between mitochondrial function and auxin by establishing a cascade of cellular events that link them through ROS formation, metabolism and root growth at different pH values.

Biosynthesis of the halogenated auxin, 4-chloroindole-3-acetic acid.

Seeds of several agriculturally important legumes are rich sources of the only halogenated plant hormone, 4-chloroindole-3-acetic acid. However, the biosynthesis of this auxin is poorly understood. Here, we show that in pea (Pisum sativum) seeds, 4-chloroindole-3-acetic acid is synthesized via the novel intermediate 4-chloroindole-3-pyruvic acid, which is produced from 4-chlorotryptophan by two aminotransferases, TRYPTOPHAN AMINOTRANSFERASE RELATED1 and TRYPTOPHAN AMINOTRANSFERASE RELATED2. We characterize a tar2 mutant, obtained by Targeting Induced Local Lesions in Genomes, the seeds of which contain dramatically reduced 4-chloroindole-3-acetic acid levels as they mature. We also show that the widespread auxin, indole-3-acetic acid, is synthesized by a parallel pathway in pea.

Reassessing the role of N-hydroxytryptamine in auxin biosynthesis.

The tryptamine pathway is one of five proposed pathways for the biosynthesis of indole-3-acetic acid (IAA), the primary auxin in plants. The enzymes AtYUC1 (Arabidopsis thaliana), FZY (Solanum lycopersicum), and ZmYUC (Zea mays) are reported to catalyze the conversion of tryptamine to N-hydroxytryptamine, putatively a rate-limiting step of the tryptamine pathway for IAA biosynthesis. This conclusion was based on in vitro assays followed by mass spectrometry or HPLC analyses. However, there are major inconsistencies between the mass spectra reported for the reaction products. Here, we present mass spectral data for authentic N-hydroxytryptamine, 5-hydroxytryptamine (serotonin), and tryptamine to demonstrate that at least some of the published mass spectral data for the YUC in vitro product are not consistent with N-hydroxytryptamine. We also show that tryptamine is not metabolized to IAA in pea (Pisum sativum) seeds, even though a PsYUC-like gene is strongly expressed in these organs. Combining these findings, we propose that at present there is insufficient evidence to consider N-hydroxytryptamine an intermediate for IAA biosynthesis.

Auxin biosynthesis in pea: characterization of the tryptamine pathway.

One pathway leading to the bioactive auxin, indole-3-acetic acid (IAA), is known as the tryptamine pathway, which is suggested to proceed in the sequence: tryptophan (Trp), tryptamine, N-hydroxytryptamine, indole-3-acetaldoxime, indole-3-acetaldehyde (IAAld), IAA. Recently, this pathway has been characterized by the YUCCA genes in Arabidopsis (Arabidopsis thaliana) and their homologs in other species. YUCCA is thought to be responsible for the conversion of tryptamine to N-hydroxytryptamine. Here we complement the genetic findings with a compound-based approach in pea (Pisum sativum), detecting potential precursors by gas chromatography/tandem-mass spectrometry. In addition, we have synthesized deuterated forms of many of the intermediates involved, and have used them to quantify the endogenous compounds, and to investigate their metabolic fates. Trp, tryptamine, IAAld, indole-3-ethanol, and IAA were detected as endogenous constituents, whereas indole-3-acetaldoxime and one of its products, indole-3-acetonitrile, were not detected. Metabolism experiments indicated that the tryptamine pathway to IAA in pea roots proceeds in the sequence: Trp, tryptamine, IAAld, IAA, with indole-3-ethanol as a side-branch product of IAAld. N-hydroxytryptamine was not detected, but we cannot exclude that it is an intermediate between tryptamine and IAAld, nor can we rule out the possibility of a Trp-independent pathway operating in pea roots.

Enzymes as Parts in Need of Replacement - and How to Extend Their Working Life.

Enzymes catalyze reactions in vivo at different rates and each enzyme molecule has a lifetime limit before it is degraded and replaced to enable catalysis to continue. Considering these rates together as a unitless ratio of catalytic cycles until replacement (CCR) provides a new quantitative tool to assess the replacement schedule of and energy investment into enzymes as they relate to function. Here, we outline the challenges of determining CCRs and new approaches to overcome them and then assess the CCRs of selected enzymes in bacteria and plants to reveal a range of seven orders of magnitude for this ratio. Modifying CCRs in plants holds promise to lower cellular costs, to tailor enzymes for particular environments, and to breed enzyme improvements for crop productivity.

Extraction, purification, methylation and GC-MS analysis of short-chain carboxylic acids for metabolic flux analysis.

Dynamic metabolic flux analysis requires efficient and effective methods for extraction, purification and analysis of a plethora of naturally-occurring compounds. One area of metabolism that would be highly informative to study using metabolic flux analysis is the tricarboxylic acid (TCA) cycle, which consists of short-chain carboxylic acids. Here, we describe a newly-developed method for extraction, purification, derivatization and analysis of short-chain carboxylic acids involved in the TCA cycle. The method consists of snap-freezing the plant material, followed by maceration and a 12-15h extraction at -80 °C. The extracts are then subject to reduction (to stabilize ß-keto acids), purified by strong anion exchange solid phase extraction and methylated with methanolic HCl. This method could also be readily adapted to quantify many other short-chain carboxylic acids.

The shifting paradigms of auxin biosynthesis.

Auxins are an important group of hormones found in all land plants and several soil-dwelling microbes. Although auxin was the first phytohormone identified, its biosynthesis remained unclear until recently. In the past few years, our understanding of auxin biosynthesis has im-proved dramatically, to the stage where many believe there is a single predominant pathway in Arabidopsis (Arabidopsis thaliana L.). However, there is still uncertainty over the applicability of these findings to other plant species. Indeed, it appears that in certain organs of some species, other pathways can operate. Here we review the key advances that have led to our current understanding of auxin biosynthesis and its many pro-posed pathways.

A mutation affecting the synthesis of 4-chloroindole-3-acetic acid.

Traditionally, schemes depicting auxin biosynthesis in plants have been notoriously complex. They have involved up to four possible pathways by which the amino acid tryptophan might be converted to the main active auxin, indole-3-acetic acid (IAA), while another pathway was suggested to bypass tryptophan altogether. It was also postulated that different plants use different pathways, further adding to the complexity. In 2011, however, it was suggested that one of the four tryptophan-dependent pathways, via indole-3-pyruvic acid (IPyA), is the main pathway in Arabidopsis thaliana, although concurrent operation of one or more other pathways has not been excluded. We recently showed that, for seeds of Pisum sativum (pea), it is possible to go one step further. Our new evidence indicates that the IPyA pathway is the only tryptophan-dependent IAA synthesis pathway operating in pea seeds. We also demonstrated that the main auxin in developing pea seeds, 4-chloroindole-3-acetic acid (4-Cl-IAA), which accumulates to levels far exceeding those of IAA, is synthesized via a chlorinated version of the IPyA pathway.

Reassessing the role of YUCCAs in auxin biosynthesis.

It is remarkable that although auxin was the first growth-promoting plant hormone to be discovered, and although more researchers work on this hormone than on any other, we cannot be definitive about the pathways of auxin synthesis in plants. In 2001, there appeared to be a dramatic development in this field, with the announcement of a new gene, and a new intermediate, purportedly from the tryptamine pathway for converting tryptophan to the main endogenous auxin, indole-3-acetic acid (IAA). Recently, however, we presented evidence challenging the original and subsequent identifications of the intermediate concerned.