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Abstract:
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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 . |