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Duck liver 'malic' enzyme. Expression in Escherichia coli and characterization of the wild-type enzyme and site-directed mutants.

ABSTRACT: A cDNA for duck liver 'malic' enzyme (EC 1.1.1.40) was subcloned into pUC-8, and the active enzyme was expressed in Escherichia coli TG-2 cells as a fusion protein including a 15-residue N-terminal leader from beta-galactosidase coded by the lacZ' gene. C99S and R70Q mutants of the enzyme were generated by the M13 mismatch technique. The recombinant enzymes were purified to near homogeneity by a simple two-step procedure and characterized relative to the enzyme isolated from duck liver. The natural duck enzyme has a subunit molecular mass of approx. 65 kDa, and the following kinetic parameters for oxidative decarboxylation of L-malate at pH 7.0: Km NADP+ (4.6 microM); Km L-malate (73 microM); kcat (160 s-1); Ka (2.4 microM) and Ka' (270 microM), dissociation constants of Mn2+ at 'tight' (activating) and 'weak' metal sites; and substrate inhibition (51% of kcat. at 8 mM-L-malate). Properties of the E. coli-derived recombinant wild-type enzyme are indistinguishable from those of the natural duck enzyme. Kinetic parameters of the R70Q mutant are relatively unaltered, indicating that Arg-70 is not required for the reaction. The C99S mutant has unchanged Km for NADP+ and parameters for the 'weak' sites (i.e. inhibition by L-malate, Ka'); however, kcat. decreased 3-fold and Km for L-malate and Ka each increased 4-fold, resulting in a catalytic efficiency [kcat./(Km NADP+ x Km L-malate x Ka)] equal to 3.7% of the natural duck enzyme. These results suggest that the positioning of Cys-99 in the sequence is important for proper binding of L-malate and bivalent metal ions.

SUBMITTER: Hsu RY

PROVIDER: S-EPMC1132620 | biostudies-other | 1992 Jun

REPOSITORIES: biostudies-other

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Project description:It is generally recognized that proteins constitute the key cellular component in shaping microbial phenotypes. Due to limited cellular resources and space, optimal allocation of proteins is crucial for microbes to facilitate maximum proliferation rates while allowing a flexible response to environmental changes. To account for the growth condition-dependent proteome in the constraint-based metabolic modeling of Escherichia coli, we consolidated a coarse-grained protein allocation approach with the explicit consideration of enzymatic constraints on reaction fluxes. Besides representing physiologically relevant wild-type phenotypes and flux distributions, the resulting protein allocation model (PAM) advances the predictability of the metabolic responses to genetic perturbations. A main driver of mutant phenotypes was ascribed to inherited regulation patterns in protein distribution among metabolic enzymes. Moreover, the PAM correctly reflected metabolic responses to an augmented protein burden imposed by the heterologous expression of green fluorescent protein. In summary, we were able to model the effects of important and frequently applied metabolic engineering approaches on microbial metabolism. Therefore, we want to promote the integration of protein allocation constraints into classical constraint-based models to foster their predictive capabilities and application for strain analysis and engineering purposes.IMPORTANCE Predictive metabolic models are important, e.g., for generating biological knowledge and designing microbes with superior performance for target compound production. Yet today's whole-cell models either show insufficient predictive capabilities or are computationally too expensive to be applied to metabolic engineering purposes. By linking the inherent genotype-phenotype relationship to a complete representation of the proteome, the PAM advances the accuracy of simulated phenotypes and intracellular flux distributions of E. coli Being equally computationally lightweight as classical stoichiometric models and allowing for the application of established in silico tools, the PAM and related simulation approaches will foster the use of a model-driven metabolic research. Applications range from the investigation of mechanisms of microbial evolution to the determination of optimal strain design strategies in metabolic engineering, thus supporting basic scientists and engineers alike.

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Duck liver 'malic' enzyme. Expression in Escherichia coli and characterization of the wild-type enzyme and site-directed mutants.

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