Characterization of the activities of trans-3-chloroacrylic acid dehalogenase and cis-3-chloroacrylic acid dehalogenase and malonate semialdehyde decarboxylase homologues : mechanism and evolutionary implications

Repository

Characterization of the activities of trans-3-chloroacrylic acid dehalogenase and cis-3-chloroacrylic acid dehalogenase and malonate semialdehyde decarboxylase homologues : mechanism and evolutionary implications

Show full record

Title: Characterization of the activities of trans-3-chloroacrylic acid dehalogenase and cis-3-chloroacrylic acid dehalogenase and malonate semialdehyde decarboxylase homologues : mechanism and evolutionary implications
Author: Serrano, Hector, doctor of pharmacy
Abstract: Members of the tautomerase superfamily are characterized by a [beta-alpha-beta] structural fold motif as well as a catalytic N-terminal proline (Pro-1). Three members of the superfamily are involved in the degradation of the nematocide 1,3-dichloropopene; trans-3-chloroacrylic acid dehalogenase (CaaD), cis-3-chloroacrylic acid dehalogenase (cis-CaaD) and malonate semialdehyde decarboxylase (MSAD). CaaD and cis-CaaD are involved in the hydration of their respective 3-chloroacrylic acid isomers to generate malonate semialdehyde. Subsequently, MSAD is responsible for catalyzing the decarboxylation of malonate semialdehyde to generate acetaldehyde. All three of these enzymes contain an N-terminal proline (Pro-1) that functions as a general acid, in contrast to other tautomerase superfamily members, such as 4-oxalocrotonate tautomerase (4-OT) and macrophage migration inhibitory factor (MIF), where Pro-1 acts as a catalytic base. Two new members of the tautomerase superfamily have been cloned and characterized; FG41 MSAD, a homologue of MSAD from Coryneform Bacterium strain FG41, and Cg10062, a homologue of cis-CaaD from Corynebacterium glutamicum, with low-level cis-CaaD and CaaD activities. As part of an effort to delineate the mechanisms of CaaD, cis-CaaD and Cg10062, secondary activities for all three enzymes were characterized. The three enzymes function as efficient phenylpyruvate tautomerases (PPT), converting phenylenolpyruvate to phenylpyruvate. The activity also indicates that the active site of these three enzymes can ketonize enol compounds, thereby providing evidence for the presence of an enediolate intermediate. The characterization of FG41 MSAD uncovered an activity it shares with MSAD. FG41 MSAD catalyzes the hydration of 2-oxo-3-pentynoate, but at a rate that is 50-fold less efficient than that of MSAD (as assessed by kcat/Km values). Mutagenesis studies of FG41 MSAD revealed that a single mutation resulted in a 8-fold increase in the activity. The characterization of Cg10062 and attempts to enhance the low-level cis-CaaD activity demonstrated the need for a bacterial screen that could screen a library of mutants. The resulting bacterial screen could be used to screen other members of the superfamily for dehalogenase activity. An in-depth exploration of the Cg10062 and FG41 MSAD activities may lead to a better understanding of the mechanism of cis-CaaD and MSAD and further delineate the evolutionary pathway for the tautomerase superfamily.
Department: Pharmacy
Subject: trans-3-chloroacrylic acid dehalogenase cis-3-chloroacrylic acid dehalogenase Malonate semialdehyde decarboxylase Tautomerase superfamily Enzymes
URI: http://hdl.handle.net/2152/17772
Date: 2009-12

Files in this work

Download File: serranoh26629.pdf
Size: 7.364Mb
Format: application/pdf

This work appears in the following Collection(s)

Show full record


Advanced Search

Browse

My Account

Statistics

Information