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Biomedical subjects

Bryan T Greenhagen

Publications and source records attributed to Bryan T Greenhagen.

6 recordsLinked to original sources
PubMed2007

Metabolic engineering of sesquiterpene metabolism in yeast.

Terpenes are structurally diverse compounds that are of interest because of their biological activities and industrial value. These compounds consist of chirally rich hydrocarbon backbones derived from terpene synthases, which are subsequently decorated with hydroxyl substituents catalyzed by terpene hydroxylases. Availability of these compounds is, however, limited by intractable synthetic means and because they are produced in low amounts and as complex mixtures by natural sources. We engineered yeast for sesquiterpene accumulation by introducing genetic modifications that enable the yeast to accumulate high levels of the key intermediate farnesyl diphosphate (FPP). Co-expression of terpene synthase genes diverted the enlarged FPP pool to greater than 80 mg/L of sesquiterpene. Efficient coupling of terpene production with hydroxylation was also demonstrated by coordinate expression of terpene hydroxylase activity, yielding 50 mg/L each of hydrocarbon and hydroxylated products. These yeast now provide a convenient format for investigating catalytic coupling between terpene synthases and hydroxylases, as well as a platform for the industrial production of high value, single-entity and stereochemically unique terpenes.

Alkyl and Aryl Transferases↗
PubMed2006

Identifying and manipulating structural determinates linking catalytic specificities in terpene synthases.

Terpene synthases are a mechanistically intriguing family of enzymes that catalyze complex, multistep reactions that are capable of generating hundreds of structurally diverse hydrocarbon and oxygenated scaffolds of biological and commercial importance. Interestingly, distantly related terpene synthases from fungi to plants all contain an invariant three-dimensional fold, and molecular comparisons of their active sites indicate that they are enriched with relatively inert amino acid residues that do not react directly with the reaction intermediates. Therefore, catalytic specificity appears to rely on the contour and dynamics of the active site created by the positioning of amino acid backbones and side chains on this catalytic surface and by supporting layers of residues surrounding the synthase active site cavity. Despite the high degree of structural relatedness among terpene synthases, previous studies suggest that no clear relationship between phylogenic organization and catalytic specificities is easily deciphered. We now report on the reciprocal interconversion of catalytic specificities between two distinct yet evolutionarily related terpene synthases based on the systematic identification and mutational replacement of variable residues within and surrounding the active site. Furthermore, we uncover previously undocumented biosynthetic activity during the interconversion, activity that could have been present in a common ancestor of these two highly related synthases. These results provide a simplified means for mapping structural features that are responsible for functional attributes and a strategy for identifying residues that differentiate divergent biosynthetic properties in phylogenetically related terpene synthases.

Alkyl and Aryl Transferases↗
PubMed2005

Surrogate splicing for functional analysis of sesquiterpene synthase genes.

A method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns is described. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product. The method was optimized by examining the expression of an intron-containing beta-glucuronidase gene agroinfiltrated into petunia (Petunia hybrida) leaves, and its utility was demonstrated by defining the function of two previously uncharacterized terpene synthases. A tobacco (Nicotiana tabacum) terpene synthase-like gene containing six predicted introns was characterized as having 5-epi-aristolochene synthase activity, while an Arabidopsis (Arabidopsis thaliana) gene previously annotated as a terpene synthase was shown to possess a novel sesquiterpene synthase activity for alpha-barbatene, thujopsene, and beta-chamigrene biosynthesis.

Alternative Splicing↗
PubMed2004

Kinetic and molecular analysis of 5-epiaristolochene 1,3-dihydroxylase, a cytochrome P450 enzyme catalyzing successive hydroxylations of sesquiterpenes.

The final step of capsidiol biosynthesis is catalyzed by 5-epiaristolochene dihydroxylase (EAH), a cytochrome P450 enzyme that catalyzes the regio- and stereospecific insertion of two hydroxyl moieties into the bicyclic sesquiterpene 5-epiaristolochene (EA). Detailed kinetic studies using EA and the two possible monohydroxylated intermediates demonstrated the release of 1beta-hydroxy-EA ((OH)EA) at high EA concentrations and a 10-fold catalytic preference for 1beta(OH)EA versus 3alpha(OH)EA, indicative of a preferred reaction order of hydroxylation at C-1, followed by that at C-3. Sequence alignments and homology modeling identified active-site residues tested for their contribution to substrate specificity and overall enzymatic activity. Mutants EAH-S368C and EAH-S368V exhibited wild-type catalytic efficiencies for 1beta(OH)EA biosynthesis, but were devoid of the successive hydroxylation activity for capsidiol biosynthesis. In contrast to EAH-S368C, EAH-S368V catalyzed the relative equal biosynthesis of 1beta(OH)EA, 2beta(OH)EA, and 3beta(OH)EA from EA with wild-type efficiency. Moreover, EAH-S368V converted approximately 1.5% of these monohydroxylated products to their respective ketone forms. Alanine and threonine mutations at position 368 were significantly compromised in their conversion rates of EA to capsidiol and correlated with 3.6- and 5.7-fold increases in their Km values for the 1beta(OH)EA intermediate, respectively. A role for Ile486 in the successive hydroxylations of EA was also suggested by the EAH-I468A mutant, which produced significant amounts 1beta(OH)EA, but negligible amounts of capsidiol from EA. The altered product profile of the EAH-I486A mutant correlated with a 3.6-fold higher Km for EA and a 4.4-fold slower turnover rate (kcat) for 1beta(OH)EA. These kinetic and mutational studies were correlated with substrate docking predictions to suggest how Ser368 and Ile486 might contribute to active-site topology, substrate binding, and substrate presentation to the oxo-Fe-heme reaction center.

Amino Acid Sequence↗
PubMed2003

Probing sesquiterpene hydroxylase activities in a coupled assay with terpene synthases.

5-epi-Aristolochene dihydroxylase (EAH) catalyzes unique stereo- and regiospecific hydroxylations of a bicyclic sesquiterpene hydrocarbon to generate capsidiol. To define functional and mechanistic features of the EAH enzyme, the utility of a coupled assay using readily available sesquiterpene synthases and microsomes from yeast overexpressing the EAH enzyme was determined. Capsidiol and deoxycapsidiol biosyntheses were readily measured in coupled assays consisting of 5-epi-aristolochene synthase and EAH as determined by the incorporation of radiolabeled farnesyl diphosphate into thin-layer chromatography-isolated products and verified by gas chromatography-mass spectrometry analysis. The assays were dependent on the amounts of synthase and hydroxylase protein added, the incubation times, and the presence of nicotinamide adenine dinucleotide phosphate. The utility of this coupled assay was extended by examining the relative efficiency of the EAH enzyme to catalyze hydroxylations of different sesquiterpene skeletons generated by other terpene synthases.

Alkyl and Aryl Transferases↗