Site-directed mutagenesis of an extradiol dioxygenase involved in tetralin biodegradation identifies residues important for activity or substrate specificity

Abstract

The sequence of the extradiol dioxygenase ThnC, involved in tetralin biodegradation, was aligned with other extradiol dioxygenases involved in biodegradation of polycyclic compounds, and a three-dimensional model of ThnC, based on the structure of the previously crystallized 2,3-dihydroxybiphenyl dioxygenase from Burkholderia fungorum LB400, was built. In order to assess the functional importance of some non-active-site residues whose relevance could not be established by structural information, a number of positions surrounding the substrate-binding site were mutated in ThnC. Ten mutant proteins were purified and their activity towards 1,2-dihydroxytetralin, 1,2-dihydroxynaphthalene and 2,3-dihydroxybiphenyl was characterized. N213H, Q198H, G206M, A282R and A282G mutants increased k cat /K m at least twofold using 1,2-dihydroxytetralin as the substrate, thus showing that activity of ThnC is not maximized for this substrate. N213H and Q198H mutants increased k cat /K m using any of the substrates tested, thus showing the relevance for activity of these two histidines, which are highly conserved in dihydroxybiphenyl dioxygenases, but not present in dihydroxynaphthalene dioxygenases. Different substitutions in position 282 had different effects on general activity or substrate specificity, thus showing the functional importance of the most C-terminal b-sheet of the protein. A251M and G206M mutants showed increased activity specifically for a particular substrate. N213H, G206M, A282R, A282G and Y177I substitutions resulted in enzymes more tolerant to acidic pH, the most striking effect being observed in mutant Y177I, which showed maximal activity at pH 5?5. In addition, Q198D and V175D mutants, which had altered K m , also showed altered sensitivity to substrate inhibition, thus indicating that inhibition is exerted through the same binding site. This mutational analysis, therefore, identified conserved residues important for activity or substrate specificity, and also shed some light on the mechanism of substrate inhibition exhibited by extradiol dioxygenases.

Difference in kinetic behaviour of catechol 2,3-dioxygenase variants from a polluted environment

“…Such a difference is of the same order of magnitude as that previously described for the site-directed mutants of an extradiol dioxygenase involved in tetralin degradation, for which turnover numbers could be increased by a factor of three compared to the wild-type (Andujar & Santero, 2003).…”

Memo is a cofilin-interacting protein that influences PLCγ1 and cofilin activities, and is essential for maintaining directionality during ErbB2-induced tumor-cell migration

“…The 2.1 Å crystal structure of Memo revealed that it is structurally homologous to a class of non-heme iron dioxygenases that are mainly found in bacteria (Andujar and Santero, 2003); however, we were unable to detect metal binding or enzymatic activity (Qiu et al, 2008). To gain more insight into Memo function, we used the yeast two-hybrid (YTH) approach to identify Memo-interacting proteins.…”

Integrated Response to Inducers by Communication between a Catabolic Pathway and Its Regulatory System

“…Genes required for tetralin biodegradation ( Fig. 1) have been sequenced, and the functions of their gene products have been characterized to elucidate the biodegradation pathway (1,2,(16)(17)(18)27). As observed for other aromatic compounds, degradation of tetralin is initiated by dioxygenation of the aromatic ring, a reaction requiring oxygen and an external electron supply, which is catalyzed by the tetralin dioxygenase enzymatic complex encoded by four thnA genes (27) (Fig.…”