A Specific, Highly Active Malate Dehydrogenase by Redesign of a Lactate Dehydrogenase Framework

Wilks, Helen M.; Hart, Keith; Feeney, R; Dunn, Cameron R.; Muirhead, H.; Chia, William; Barstow, David A.; Atkinson, Tony; Clarke, Anthony R.; Holbrook, J. John

doi:10.1126/science.3201242

Cited by 282 publications

(192 citation statements)

References 25 publications

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“…An R102Q mutant enzyme of E. coli MDH had the effect of relaxing the high degree of specificity for the substrate oxaloacetate (Nicholls et al, 1992). These results were different from those for a Q102R mutant of B. stearothermophilus LDH where MDH specificity was conferred on the LDH enzyme framework (Wilks et al, 1988) and for an R102Q mutant of H . marismortui MDH where LDH activity was conferred on the MDH enzyme framework (Cendrin et al, 1993) (Fig.…”

Section: Modulation Of Substrate Specificitycontrasting

confidence: 88%

Malate dehydrogenase: A model for structure, evolution, and catalysis

1994

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Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3-dimensional structures of several malate dehydrogenases are similar, despite their low amino acid sequence identity. The coenzyme specificity of malate dehydrogenase may be modulated by substitution of a single residue, as can the substrate specificity. The mechanism of catalysis of malate dehydrogenase is similar to that of lactate dehydrogenase, an enzyme with which it shares a similar 3-dimensional structure. Substitution of a single amino acid residue of a lactate dehydrogenase changes the enzyme specificity to that of a malate dehydrogenase, but a similar substitution in a malate dehydrogenase resulted in relaxation of the high degree of specificity for oxaloacetate. Knowledge of the 3-dimensional structures of malate and lactate dehydrogenases allows the redesign of enzymes by rational rather than random mutation and may have important commercial implications.Keywords: malate dehydrogenase; molecular evolution; protein engineering Many industrial processes involve chemical conversions of organic compounds where productivity is achieved by use of relatively nonspecific inorganic catalysts. Recent advances in the biological sciences have enhanced the potential for use of enzyme catalysts, which offer a number of advantages over conventional chemical catalysts, including high specificity for the reactants, improved reaction rates, and the use of mild operational conditions. There are already many examples of commercial applications where the properties of natural enzyme catalysts have been exploited to perform synthesis and degradation of compounds, as diagnostic reagents and in research applications. Enzymes isolated from natural sources have been optimized through evolution to perform a particular biological role and often have disadvantages for the design of specific chemical application protocols. Genetic manipulation via protein engineering allows the rational redesign of an enzyme primary sequence to change its chemical and physical properties. A lack of structural information for many enzymes means that the physiological effects of amino acid sequence alterations are difficult to predict. The enzyme malate dehydrogenase (MDH; EC 1.1.1.37) is a good candidate to test the efficiency of predictive amino acid

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Section: Modulation Of Substrate Specificitycontrasting

confidence: 88%

Malate dehydrogenase: A model for structure, evolution, and catalysis

1994

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“…5a). Prior reports demonstrated that the corresponding residue in bacterial LDH plays an important role in determining substrate specificity 39,40 .…”

Section: Resultsmentioning

confidence: 99%

“…Indeed, mutation of the corresponding residue in bacterial LDH from glutamine to arginine (Q102R) enhanced the ability of the enzyme to reduce oxaloacetate 39,40 . Therefore, we generated vectors expressing either wildtype LDHA (LDHA WT) or LDHA with a point mutation resulting in replacement of glutamine 100 with arginine (LDHA Q100R).…”

Section: Resultsmentioning

confidence: 99%

L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH

et al. 2017

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The metabolite 2-hydroxyglutarate (2HG) can be produced as either a D(R)- or L(S)- enantiomer, each of which inhibits alpha-ketoglutarate (αKG)-dependent enzymes involved in diverse biologic processes. Oncogenic mutations in isocitrate dehydrogenase produce D-2HG, which causes a pathologic blockade in cell differentiation. On the other hand, oxygen limitation leads to accumulation of L-2HG, which can facilitate physiologic adaptation to hypoxic stress in both normal and malignant cells. Here we demonstrate that purified lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) catalyze stereospecific production of L-2HG via ‘promiscuous’ reduction of the alternative substrate αKG. Acidic pH enhances production of L-2HG by promoting a protonated form of αKG that binds to a key residue in the substrate-binding pocket of LDHA. Acid-enhanced production of L-2HG leads to stabilization of hypoxia-inducible factor 1 alpha (HIF-1α) in normoxia. These findings offer insights into mechanisms whereby microenvironmental factors influence production of metabolites that alter cell fate and function.

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“…In the case of malate dehydrogenase, a single point mutation was sufficient to convert it into a lactate dehydrogenase, with a k cat /K m shift of 10 7 (40). However, for many systems, larger numbers of mutations or more drastic changes are required to alter specificity (41,42).…”

Section: Table 2 Kinetic Parameters Measured For Sortase Mutantsmentioning

confidence: 99%

Engineering the Substrate Specificity of Staphylococcus aureus Sortase A

Bentley

Gaweska

Kielec

et al. 2007

Journal of Biological Chemistry

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The Staphylococcus aureus transpeptidase Sortase A (SrtA) anchors virulence and colonization-associated surface proteins to the cell wall. SrtA selectively recognizes a C-terminal LPXTG motif, whereas the related transpeptidase Sortase B (SrtB) recognizes a C-terminal NPQTN motif. In both enzymes, cleavage occurs after the conserved threonine, followed by amide bond formation between threonine and the pentaglycine cross-bridge of cell wall peptidoglycan. Genetic and biochemical studies strongly suggest that SrtA and SrtB exhibit exquisite specificity for their recognition motifs. To better understand the origins of substrate specificity within these two isoforms, we used sequence and structural analysis to predict residues and domains likely to be involved in conferring substrate specificity. Mutational analyses and domain swapping experiments were conducted to test their function in substrate recognition and specificity. Marked changes in the specificity profile of SrtA were obtained by replacing the ␤6/␤7 loop in SrtA with the corresponding domain from SrtB. The chimeric ␤6/␤7 loop swap enzyme (SrtLS) conferred the ability to acylate NPQTNcontaining substrates, with a k cat /K m app of 0.0062 ؎ 0.003 M ؊1 s ؊1 . This enzyme was unable to perform the transpeptidation stage of the reaction, suggesting that additional domains are required for transpeptidation to occur. The overall catalytic specificity profile (k cat /K m app (NPQTN)/k cat /K m app (LPETG)) of SrtLS was altered 700,000-fold from SrtA. These results indicate that the ␤6/␤7 loop is an important site for substrate recognition in sortases.

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A Specific, Highly Active Malate Dehydrogenase by Redesign of a Lactate Dehydrogenase Framework

Cited by 282 publications

References 25 publications

Malate dehydrogenase: A model for structure, evolution, and catalysis

Malate dehydrogenase: A model for structure, evolution, and catalysis

L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH

Engineering the Substrate Specificity of Staphylococcus aureus Sortase A

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