Browsing by Subject "Bi-functional"

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    Identification of pyruvate decarboxylase/indole pyruvate decarboxylase gene family members from Arabidopsis thaliana.
    (2009-09) Ye, Songqing
    Several biologically important and diverse reactions are regulated by thiamine pyrophosphate (TPP) cofactor-dependent metabolic enzymes, including pyruvate decarboxylase (PDC), indole pyruvate decarboxylase (IPDC), and acetohydroxy acid synthase. PDC is a critical enzyme in plant metabolism that regulates energy production especially during periods of anaerobic stress. IPDC has long been proposed as a key enzyme in the biosynthesis of the plant hormone indole-3-acetic acid (IAA) from tryptophan. Six putative Arabidopsis thaliana PDC gene family members have been individually cloned and expressed in E. coli, and recombinant PDC proteins were purified and biochemically characterized. AtPDC2 was identified as a unique functional PDC based on its measured biochemical activity. The pH and temperature optima for the recombinant protein were 6.2 and 55°C, respectively, and the Km was 3.5 mM. Also, addition of 0.5 mM TPP and 5 mM Mg2+ resulted in the highest activity. However, AtPDC2 lacked any measurable IPDC activity as determined by gas chromatography-mass spectrometry (GC-MS)-based methods. Thus, this mono-functional PDC was different from the more thoroughly studied microbial PDCs, which all have bi-functional activity toward both indole-3-pyruvate and pyruvate substrates. These findings suggest a potential regulatory role for the catalytically inactive PDC proteins in modulation of PDC activity, similar to a mechanism proposed for yeast. None of the pdc mutants showed a change in resistance to chlorsulfuron or imazamox herbicides, and this result was also consistent with the hypothesis that the inactive AtPDC genes may play a role in PDC activity regulation in Arabidopsis. Studies presented here show that the genes most likely to encode proteins with PDC activity or IPDC activity, the PDC gene family, all lack IPDC activity and all except one lack PDC activity. Furthermore, all Arabidopis PDC T-DNA insertion mutants were found to share the same shade avoidance phenotype to as did wild-type plants. These findings bring into question the physiological significance of the IPA pathway for auxin biosynthesis as has been previously proposed. Very low levels of IPDC activity are difficult to measure using procedures developed for the enzyme activity of proteins from bacteria, which produce substantial levels of indole acetaldehyde (IAAld) from indole-3-pyruvate (IPA). To determine the potential activity of plant enzymes, either expressed in E. coli or extracted from Arabidopsis plants, GC-MS assay methods were developed with high sensitivity and specificity. For expressed proteins, IAAld produced from IPA was measured directly using indole carboxaldehyde as an internal standard. This procedure failed, however, to detect IPA in the presence of plant protein extracts; thus, a coupled in vitro reaction with aldehyde dehydrogenase that produced IAA from IPA was developed, and the IAA was quantified using [13C6]IAA as an internal standard, methylation with diazomethane, and GC-MS detection. Together, these methods provide important sensitive and precise methods for the search for IPDC activity in the plant kingdom.