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

Keri L Colabroy; Ian R Smith; Alexander H S Vlahos; Androo J Markham; Matthew E Jakubik

doi:10.1016/j.bbapap.2013.12.005

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

Biochim Biophys Acta. 2014 Mar;1844(3):607-14. doi: 10.1016/j.bbapap.2013.12.005. Epub 2013 Dec 22.

Authors

Keri L Colabroy¹, Ian R Smith², Alexander H S Vlahos², Androo J Markham², Matthew E Jakubik²

Affiliations

¹ Department of Chemistry, Muhlenberg College, Allentown, PA 18104, USA. Electronic address: colabroy@muhlenberg.edu.
² Department of Chemistry, Muhlenberg College, Allentown, PA 18104, USA.

PMID: 24365644
DOI: 10.1016/j.bbapap.2013.12.005

Abstract

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.

Keywords: Extradiol dioxygenase; Global fitting; KinTek Explorer; Kinetic mechanism.

MeSH terms

Kinetics
Oxygenases / chemistry*
Streptomyces / enzymology*

Substances

Oxygenases
extradiol dioxygenase