Purification and characterization of the malate dehydrogenase from Streptomyces aureofaciens

Mikulasova, D

doi:10.1016/s0378-1097(97)00567-3

Cited by 3 publications

(1 citation statement)

References 19 publications

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“…This is higher than what has been observed with the hyperthermophilic Bacillus candolyticus, which lost 50% of its activity after a 1-min incubation at 59°C, but similar to the values for Vulcanithermus medioatlanticus, which retained over 75% activity after 15 min of incubation at 60°C (30,32). The K m for OAA was significantly higher than for many characterized MDHs, such as the ones from V. medioatlanticus (0.048 mM), Corynebacterium glutamicum (0.057 mM), and Streptomyces aureofaciens (0.1 mM) (30,33,34 (33,38,39).…”

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confidence: 68%

Reassessment of the Transhydrogenase/Malate Shunt Pathway in Clostridium thermocellum ATCC 27405 through Kinetic Characterization of Malic Enzyme and Malate Dehydrogenase

Taillefer

Rydzak

Levin

et al. 2015

Appl Environ Microbiol

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Clostridium thermocellum produces ethanol as one of its major end products from direct fermentation of cellulosic biomass. Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing. However, a better understanding of the metabolic pathways, along with their putative regulation, could lead to improved strategies for increasing the production of ethanol. In the absence of an annotated pyruvate kinase in the genome, alternate means of generating pyruvate have been sought. Previous proteomic and transcriptomic work detected high levels of a malate dehydrogenase and malic enzyme, which may be used as part of a malate shunt for the generation of pyruvate from phosphoenolpyruvate. The purification and characterization of the malate dehydrogenase and malic enzyme are described in order to elucidate their putative roles in malate shunt and their potential role in C. thermocellum metabolism. The malate dehydrogenase catalyzed the reduction of oxaloacetate to malate utilizing NADH or NADPH with a k cat of 45.8 s ؊1 or 14.9 s ؊1 , respectively, resulting in a 12-fold increase in catalytic efficiency when using NADH over NADPH. The malic enzyme displayed reversible malate decarboxylation activity with a k cat of 520.8 s ؊1 . The malic enzyme used NADP ؉ as a cofactor along with NH 4 ؉ and Mn 2؉ as activators. Pyrophosphate was found to be a potent inhibitor of malic enzyme activity, with a K i of 0.036 mM. We propose a putative regulatory mechanism of the malate shunt by pyrophosphate and NH 4 ؉ based on the characterization of the malate dehydrogenase and malic enzyme.C lostridium thermocellum is a Gram-positive, anaerobic thermophile capable of reaching one of the highest growth rates on crystalline cellulose (1, 2). Furthermore, given its native ability to produce ethanol and H 2 , C. thermocellum is seen as an attractive microorganism for the production of biofuels via consolidated bioprocessing of lignocellulosic biomass. Unfortunately, current yields and production rates of ethanol and/or H 2 are low due to branched product pathways (3-5) which redirect carbon and electron flux away from a desired biofuel. These unwanted products include lactate, formate, and/or acetate (6-8), as well as secreted amino acids (9, 10). Thus, redirecting carbon and electron flux away from these secreted products toward either ethanol or H 2 may improve the economic viability of biofuels production using C. thermocellum.A key pathway node involved in the interconversion of phosphoenolpyruvate (PEP) and pyruvate, which may catalyze either substrate level phosphorylation or transhydrogenation reactions between NADH to NADP ϩ , has been revisited with C. thermocellum (10, 11). The interconversion of PEP and pyruvate may be catalyzed using a number of different putative enzymes. In contrast to most clostridia and other fermentative ethanol and/or H 2 -producing organisms (7), C. thermocellum ATCC 27405 (GenBank accession number NC_009012.1) does not encode a pyruvate kinase, which catalyzes PE...

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Section: Discussionsupporting

confidence: 68%

Reassessment of the Transhydrogenase/Malate Shunt Pathway in Clostridium thermocellum ATCC 27405 through Kinetic Characterization of Malic Enzyme and Malate Dehydrogenase

Taillefer

Rydzak

Levin

et al. 2015

Appl Environ Microbiol

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Identification and Biochemical Characterization of a Thermostable Malate Dehydrogenase from the MesophileStreptomyces coelicolorA3(2)

Cao

Wang

et al. 2010

Bioscience, Biotechnology, and Biochemistry

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Purification and Characterisation of Malate Dehydrogenase From Synechocystis sp. PCC 6803: Biochemical Barrier of the Oxidative Tricarboxylic Acid Cycle

et al. 2018

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Cyanobacteria possess an atypical tricarboxylic acid (TCA) cycle with various bypasses. Previous studies have suggested that a cyclic flow through the TCA cycle is not essential for cyanobacteria under normal growth conditions. The cyanobacterial TCA cycle is, thus, different from that in other bacteria, and the biochemical properties of enzymes in this TCA cycle are less understood. In this study, we reveal the biochemical characteristics of malate dehydrogenase (MDH) from Synechocystis sp. PCC 6803 MDH (SyMDH). The optimal temperature of SyMDH activity was 45–50°C and SyMDH was more thermostable than MDHs from other mesophilic microorganisms. The optimal pH of SyMDH varied with the direction of the reaction: pH 8.0 for the oxidative reaction and pH 6.5 for the reductive reaction. The reductive reaction catalysed by SyMDH was activated by magnesium ions and fumarate, indicating that SyMDH is regulated by a positive feedback mechanism. The Km-value of SyMDH for malate was approximately 210-fold higher than that for oxaloacetate and the Km-value for NAD+ was approximately 19-fold higher than that for NADH. The catalytic efficiency of SyMDH for the reductive reaction, deduced from kcat-values, was also higher than that for the oxidative reaction. These results indicate that SyMDH is more efficient in the reductive reaction in the TCA cycle, and it plays key roles in determining the direction of the TCA cycle in this cyanobacterium.

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Purification and characterization of the malate dehydrogenase from Streptomyces aureofaciens

Cited by 3 publications

References 19 publications

Reassessment of the Transhydrogenase/Malate Shunt Pathway in Clostridium thermocellum ATCC 27405 through Kinetic Characterization of Malic Enzyme and Malate Dehydrogenase

Reassessment of the Transhydrogenase/Malate Shunt Pathway in Clostridium thermocellum ATCC 27405 through Kinetic Characterization of Malic Enzyme and Malate Dehydrogenase

Identification and Biochemical Characterization of a Thermostable Malate Dehydrogenase from the MesophileStreptomyces coelicolorA3(2)

Purification and Characterisation of Malate Dehydrogenase From Synechocystis sp. PCC 6803: Biochemical Barrier of the Oxidative Tricarboxylic Acid Cycle

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