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Rational redesign of the 4-chlorobenzoate binding site of 4-chlorobenzoate: coenzyme a ligase for expanded substrate range.

ABSTRACT: Environmental aromatic acids are transformed to chemical energy in bacteria that possess the requisite secondary pathways. Some of these pathways rely on the activation of the aromatic acid by coenzyme A (CoA) thioesterification catalyzed by an aromatic acid: CoA ligase. Adaptation of such pathways to the bioremediation of man-made pollutants such as polychlorinated biphenyl (PCB) and dichlorodiphenyltrichloroethane (DDT) requires that the chlorinated benzoic acid byproduct that is formed be able to be eliminated by further degradation. To take advantage of natural benzoic acid degrading pathways requiring initial ring activation by thioesterification, the pathway aromatic acid:CoA ligase must be an effective catalyst with the chlorinated benzoic acid. This study, which focuses on the 4-chlorobenzoate:CoA ligase (CBL) of the 4-monochlorobiphenyl degrading bacterium Alcaligenes sp. strain ALP83, was carried out to determine if the 4-chlorobenzoate binding site of this enzyme can be transformed by rational design to recognize the chlorobenzoic acids formed in the course of breakdown of other environmental PCB congeners. The fundamental question addressed in this study is whether it is possible to add or subtract space from the substrate-binding pocket of this ligase (to complement the topology of the unnatural aromatic substrate) without causing disruption of the ligase catalytic machinery. Herein, we report the results of a substrate specificity analysis that, when interpreted within the context of the X-ray crystal structures, set the stage for the rational design of the ligase for thioesterification of two PCB-derived chlorobenzoic acids. The ligase was first optimized to catalyze CoA thioesterification of 3,4-dichlorobenzoic acid, a poor substrate, by truncating Ile303, a large hydrophobic residue that packs against the ring meta-C(H) group. The structural basis for the approximately 100-fold enhancement in the rate of 3,4-dichlorobenzoate thioesterification catalyzed by the I303A and I303G CBL mutants was validated by determination of the crystal structure of the 3,4-dichlorobenzoate-bound enzymes. Determinations of the structures of I303 mutant complexes of 3-chlorobenzoate, a very poor substrate, revealed nonproductive binding as a result of the inability of the substrate ring C(4)H group to fill the pocket that binds the C(4)Cl group of the native substrate. The C(4)Cl pocket of the CBL I303A mutant was then reduced in size by strategic amino acid replacement. A 54-fold improvement in catalytic efficiency was observed for the CBL F184W/I303A/V209T triple mutant. The results of this investigation are interpreted as evidence that the plasticity of the ligase catalytic scaffold is sufficient to allow expansion of substrate range by rational design. The combination of structural and kinetic analyses of the constructed mutants proved to be an effective approach to engineering the ligase for novel substrates.

SUBMITTER: Wu R

PROVIDER: S-EPMC4710512 | biostudies-literature | 2007 Dec

REPOSITORIES: biostudies-literature

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Rational redesign of the 4-chlorobenzoate binding site of 4-chlorobenzoate: coenzyme a ligase for expanded substrate range.

Wu Rui R Reger Albert S AS Cao Jian J Gulick Andrew M AM Dunaway-Mariano Debra D

Biochemistry 20071121 50

Environmental aromatic acids are transformed to chemical energy in bacteria that possess the requisite secondary pathways. Some of these pathways rely on the activation of the aromatic acid by coenzyme A (CoA) thioesterification catalyzed by an aromatic acid: CoA ligase. Adaptation of such pathways to the bioremediation of man-made pollutants such as polychlorinated biphenyl (PCB) and dichlorodiphenyltrichloroethane (DDT) requires that the chlorinated benzoic acid byproduct that is formed be abl ...[more]

PMID: 18027984

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Project description:Within the accompanying paper in this issue (Reger et al. (2008) Biochemistry, 47, 8016-8025) we reported the X-ray structure of 4-chlorobenzoate:CoA ligase (CBL) bound with 4-chlorobenzoyl-adenylate (4-CB-AMP) and the X-ray structure of CBL bound with 4-chlorophenacyl-CoA (4-CP-CoA) (an inert analogue of the product 4-chlorobenzoyl-coenzyme A (4-CB-CoA)) and AMP. These structures defined two CBL conformational states. In conformation 1, CBL is poised to catalyze the adenylation of 4-chlorobenzoate (4-CB) with ATP (partial reaction 1), and in conformation 2, CBL is poised to catalyze the formation of 4-CB-CoA from 4-CB-AMP and CoA (partial reaction 2). These two structures showed that, by switching from conformation 1 to conformation 2, the cap domain rotates about the domain linker and thereby changes its interface with the N-terminal domain. The present work was carried out to determine the contributions made by each of the active site residues in substrate/cofactor binding and catalysis, and also to test the role of domain alternation in catalysis. In this paper, we report the results of steady-state kinetic and transient state kinetic analysis of wild-type CBL and of a series of site-directed CBL active site mutants. The major findings are as follows. First, wild-type CBL is activated by Mg (2+) (a 12-75-fold increase in activity is observed depending on assay conditions) and its kinetic mechanism (ping-pong) supports the structure-derived prediction that PP i dissociation must precede the switch from conformation 1 to conformation 2 and therefore CoA binding. Also, transient kinetic analysis of wild-type CBL identified the rate-limiting step of the catalyzed reaction as one that follows the formation of 4-CB-CoA (viz. CBL conformational change and/or product dissociation). The single turnover rate of 4-CB and ATP to form 4-CB-AMP and PP i ( k = 300 s (-1)) is not affected by the presence of CoA, and it is approximately 3-fold faster than the turnover rate of 4-CB-AMP and CoA to form 4-CB-CoA and AMP ( k = 120 s (-1)). Second, the active site mutants screened via steady-state kinetic analysis were ranked based on the degree of reduction observed in any one of the substrate k cat/ K m values, and those scoring higher than a 50-fold reduction in k cat/ K m value were selected for further evaluation via transient state kinetic analysis. The single-turnover time courses, measured for the first partial reaction, and then for the full reaction, were analyzed to define the microscopic rate constants for the adenylation reaction and the thioesterification reaction. On the basis of our findings we propose a catalytic mechanism that centers on a small group of key residues (some of which serve in more than one role) and that includes several residues that function in domain alternation.

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Rational redesign of the 4-chlorobenzoate binding site of 4-chlorobenzoate: coenzyme a ligase for expanded substrate range.

Publications

Rational redesign of the 4-chlorobenzoate binding site of 4-chlorobenzoate: coenzyme a ligase for expanded substrate range.

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