2001 Annual Science Report
University of Colorado, Boulder Reporting | JUL 2000 – JUN 2001
Toward a Molecular Phylogeny of a Metabolic Enzyme, Maleylacetoacetate Isomerase
Toward a Molecular Phylogeny of a Metabolic Enzyme, Maleylacetoacetate Isomerase (dm)
Maleylacetoacetate (MAA) isomerase is an enzyme involved in degradation of phenylalanine and tyrosine and certain other aromatic compounds. This enzyme provides an ideal model system for studies of the evolution of metabolic pathways and the spread of metabolic genes because of its puzzling phylogenetic distribution. Glutathione-dependent MAA isomerases occur in some proteobacteria, some plants, a nematode, mice and humans. In many other organisms (the Archaea, Drosophila, other proteobacteria), MAA isomerase genes cannot be identified in genome databases. It is not clear whether the distribution of the enzyme is patchy, as might be expected if the gene has been distributed by infrequent lateral transfer events or lost selectively in certain lineages, or whether the genes in these organisms are very dissimilar and are simply not recognized by search algorithms.
Our initial efforts have been directed toward identifying additional MAA isomerases in the unfinished microbial genome databases and developing a genetic system that will allow us to easily identify MAA isomerase genes from a variety of organisms. We have identified the MAA isomerase gene in Pseudomonas putida strain KT2440 and are working on knocking it out using pKnock. The knock out strain will be used for cloning MAA isomerase genes from other microorganisms.
We have also made progress on developing a convenient and quantitative assay for MAA isomerase activity. We have amplified a homogentisate dioxygenase gene and are working on cloning it into pQE30 (a vector that adds a His6-tag to the N-terminus of the protein). This protein will be used to prepare MAA from homogentisate. We have also amplified the gene for fumarylacetoacetate hydrolase from P. putida and are working on cloning it into pQE30 for expression in E. coli. This enzyme will be used to couple the isomerization of MAA to fumarylacetoacetate to the hydrolysis of fumarylacetoacetate to fumarate and acetoacetate (two stable products). This procedure will allow us to use a continuous UV/vis assay to quantitate MAA isomerase activity in proteins from various sources.
PROJECT MEMBERS:Shelley Copley
RELATED OBJECTIVES:Objective 4.0
Expand and interpret the genomic database of a select group of key microorganisms in order to reveal the history and dynamics of evolution.
Define how ecophysiological processes structure microbial communities, influence their adaptation and evolution, and affect their detection on other planets.