L-DOPA 2,3 dioxygenase is a single domain, type I extradiol dioxygenase in the biosynthesis of the streptomycete antibiotic lincomycin. L-DOPA 2,3 dioxygenase catalyzes the oxidative cleavage of L-DOPA to form a-hydroxymuconic 6-semialdehyde (AHMS), which then cyclizes into 3-carboxy-3-hydroxyallylidene-3,4-dihydropyrrole-2-carboxylic acid (CHAPCA). The lack of active site conservation between the smaller single-domain extradiol dioxygenase and their well-studied two-domain counterparts implies that the two subfamilies operate via distinct kinetic mechanisms. In order to characterize the kinetic mechanism of the single domain L-DOPA 2,3 dioxygenase, pre-steady state and steady state experiments were combined in a global kinetic analysis using KinTek Explorer to define and constrain a kinetic model. The data were best described by a model consisting of four steps: three enzyme mediated steps followed by a nonenzymatic, acid-catalyzed cyclization of AHMS to CHAPCA. Steady state constants were determined independently by Michaelis-Menten analysis and KinTek Explorer global fitting of progress curves. Steady state constants were found to compliment the constrained microscopic rates determined by global simulation of the four step model.

Defining a kinetic mechanism for L-DOPA 2,3-dioxygenase

247th National Meeting (2014)

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