Keri L. Colabroy

Tryptophan Catabolism: Identification and Characterization of a New Degradative Pathway

A new tryptophan catabolic pathway is characterized from Burkholderia cepacia J2315. In this pathway, tryptophan is converted to 2-amino-3-carboxymuconate semialdehyde, which is enzymatically degraded to pyruvate and acetate via the intermediates 2-aminomuconate and 4-oxalocrotonate. This pathway differs from the proposed mammalian pathway which converts 2-aminomuconate to 2-ketoadipate and, ultimately, glutaryl-coenzyme A.

Biochemical characterization of L-DOPA 2,3-dioxygenase, a single-domain type I extradiol dioxygenase from lincomycin biosynthesis.

L-DOPA-2,3-dioxygenase from Streptomyces lincolnensis is a single-domain type I extradiol dioxygenase of the vicinal oxygen chelate superfamily and catalyzes the second step in the metabolism of tyrosine to the propylhygric acid moiety of the antibiotic, lincomycin. S. lincolnensis L-DOPA-2,3-dioxygenase was overexpressed, purified and reconstituted with Fe(II). The activity of L-DOPA-2,3-dioxygenase was kinetically characterized with L-DOPA (K(M)=38 microM, k(cat)=4.2 min(-1)) and additional catecholic substrates including dopamine, 3,4-dihydroxyhydrocinnamic acid, catechol and D-DOPA. 3,4-Dihydroxyphenylacetic acid was characterized as a competitive inhibitor of the enzyme (K(i) =2.2 mM). Site-directed mutagenesis and its effects on enzymatic activity were used to identify His14 and His70 as iron ligands.

A Writing-Intensive, Methods-Based Laboratory Course for Undergraduates.

Engaging undergraduate students in designing and executing original research should not only be accompanied by technique training but also intentional instruction in the critical analysis and writing of scientific literature. The course described here takes a rigorous approach to scientific reading and writing using primary literature as the model while simultaneously integrating laboratory instruction on basic enzyme purification and characterization, followed by 6 weeks of laboratory dedicated to student‐designed original research projects. In the preparation and execution of their original projects, students engage in analysis of the primary literature, proposal writing, peer review, manuscript preparation, and oral presentation. The result is a comprehensive and challenging course that teaches third‐ and fourth‐year undergraduates what it means to “think and work like a scientist.” Biochemistry and Molecular Biology Education Vol. 39, No. 3, pp. 196–203, 2011

Defining a kinetic mechanism for l-DOPA 2,3 dioxygenase, a single-domain type I extradiol dioxygenase from Streptomyces lincolnensis.

l-DOPA-2,3-dioxygenase from Streptomyces lincolnensis is a single domain type I extradiol dioxygenase of the vicinal oxygen chelate superfamily and catalyzes the second step in the metabolism of the propylhygric acid moiety of the antibiotic, lincomycin. In this report, the kinetic mechanism of l-DOPA dioxygenase is interrogated using stopped-flow in order to determine microscopic rate constants. Pre-steady state, progress curve and steady-state data were combined in a global kinetic analysis using KinTek Explorer in order to define and constrain a kinetic model for the type I l-DOPA dioxygenase. The data are best described by a four step mechanism, in which the cyclization of the enzymatic product is not enzyme catalyzed.

Tearing down to build up: Metalloenzymes in the biosynthesis lincomycin, hormaomycin and the pyrrolo [1,4]benzodiazepines.

The metabolic pathways for the production of lincomycin, hormaomycin and the antitumor pyrrolo [1,4] benzodiazepines share a vinyl substituted pyrroline carboxylic acid (3-vinyl-2,3-pyrroline-5-carboxylic acid, VPCA) as a common intermediate. Biosynthesis of this vinyl substituted pyrroline carboxylic acid intermediate requires a short, three-enzyme pathway containing two metalloenzymes: a heme-dependent l-tyrosine hydroxylase and a non-heme Fe(2+) dependent l-DOPA dioxygenase. The l-tyrosine hydroxylase is an unprecedented type of peroxidase that specifically monohydroxylates tyrosine, while the l-DOPA extradiol cleaving enzyme is a single-domain vicinal-oxygen-chelate (VOC) dioxygenase. The dioxygenase product subsequently undergoes an, as yet uncharacterized, C-C bond cleavage reaction. This mini-pathway demonstrates the use of metal-dependent chemistry typically associated with natural product degradation in order to build a compact, functionalized building block for larger, bioactive molecules.