Biomedical subjects

John M Humphreys

Publications and source records attributed to John M Humphreys.

4 recordsLinked to original sources

PubMed2004

Immunodetection and quantification of cytochromes P450 using epitope tagging: immunological, spectroscopic, and kinetic analysis of cinnamate 4-hydroxylase.

Cytochrome P450-dependent monooxygenases (P450s) are integral membrane proteins typically expressed at low levels both in vivo and by heterologous expression systems, often making quantification of these enzymes challenging. Since the time of their discovery, P450s have typically been quantified by their carbon monoxide (CO) difference spectra. Although this technique is reliable, it requires quantities of enzyme that are sometimes difficult to obtain, and spectroscopic instruments and expertise frequently unavailable in laboratories whose primary focus is genetics or molecular biology. We have developed a method for quantifying recombinant FLAG epitope-tagged proteins using fluorescence detection of a chromophore-labeled anti-FLAG monoclonal antibody and well-established immunoblot technology. The utility of this technique was tested using cinnamate 4-hydroxylase (C4H), one of the best-studied plant P450s. No substantial differences in the stability or kinetic properties were observed between the native and FLAG-tagged enzymes. Immunochemical quantification of epitope-tagged C4H reported slightly lower P450 concentrations than conventional methods but has a limit of quantification 400-fold lower than carbon monoxide difference spectroscopy.

Catalysis↗

PubMed2002

Rewriting the lignin roadmap.

Considerable interest in lignin biosynthesis has been fueled by the many roles that lignin plays in development and in resistance to biotic and abiotic stress, as well as its importance to industry and agriculture. Although the pathway leading to the lignin polymer has been studied for decades, new insights into the enzymes of the pathway have required a complete re-evaluation of how we think lignin precursors are synthesized. Although free hydroxycinnamic acids have long been thought to be key intermediates, it has become apparent that many of the hydroxylation and methylation steps in the pathway occur instead at the level of hydroxycinnamic acid esters, and their corresponding aldehydes and alcohols.

Caffeic Acids↗

PubMed2002

The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism.

The activity of p-coumarate 3-hydroxylase (C3H) is thought to be essential for the biosynthesis of lignin and many other phenylpropanoid pathway products in plants; however, no conditions suitable for the unambiguous assay of the enzyme are known. As a result, all attempts to purify the protein and clone its corresponding gene have failed. By screening for plants that accumulate reduced levels of soluble fluorescent phenylpropanoid secondary metabolites, we have identified a number of Arabidopsis mutants that display a reduced epidermal fluorescence (ref) phenotype. Using radiotracer-feeding experiments, we have determined that the ref8 mutant is unable to synthesize caffeic acid, suggesting that the mutant is defective in a gene required for the activity or expression of C3H. We have isolated the REF8 gene using positional cloning methods, and have verified that it encodes C3H by expression of the wild-type gene in yeast. Although many previous reports in the literature have suggested that C3H is a phenolase, the isolation of the REF8 gene demonstrates that the enzyme is actually a cytochrome P450-dependent monooxygenase. Although the enzyme accepts p-coumarate as a substrate, it also exhibits significant activity towards other p-hydroxylated substrates. These data may explain the previous difficulties in identifying C3H activity in plant extracts and they indicate that the currently accepted version of the lignin biosynthetic pathway is likely to be incorrect.

Arabidopsis↗

PubMed2002

Changes in secondary metabolism and deposition of an unusual lignin in the ref8 mutant of Arabidopsis.

The end products of the phenylpropanoid pathway play important roles in plant structure and development, as well as in plant defense mechanisms against biotic and abiotic stresses. From a human perspective, phenylpropanoid pathway-derived metabolites influence both human health and the potential utility of plants in agricultural contexts. The last known enzyme of the phenylpropanoid pathway that has not been characterized is p-coumarate 3-hydroxylase (C3H). By screening for plants that fail to accumulate soluble fluorescent phenylpropanoid secondary metabolites, we have identified a number of Arabidopsis mutants that display a reduced epidermal fluorescence (ref) phenotype. We have now shown that the ref8 mutant is defective in the gene encoding C3H. Phenotypic characterization of the ref8 mutant has revealed that the lack of C3H activity in the mutant leads to diverse changes in phenylpropanoid metabolism. The ref8 mutant accumulates p-coumarate esters in place of the sinapoylmalate found in wild-type plants. The mutant also deposits a lignin formed primarily from p-coumaryl alcohol, a monomer that is at best a minor component in the lignin of other plants. Finally, the mutant displays developmental defects and is subject to fungal attack, suggesting that phenylpropanoid pathway products downstream of REF8 may be required for normal plant development and disease resistance.

Arabidopsis↗