Alteration of coenzyme specificity of malate dehydrogenase from Streptomyces coelicolor A3(2) by site-directed mutagenesis

Ge, Yadong; Song, Ping; Cao, Zhengyu; Wang, P.; Zhu, Guoping

doi:10.4238/2014.july.29.3

Cited by 6 publications

(5 citation statements)

References 23 publications

(34 reference statements)

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“…Serine has been targeted to change coenzyme specificity toward NAD, and incorporated in the other direction (Figures 1E,F ). Ser usually interacts with the phosphate group of NADP stabilizing the coenzyme binding (Schepens et al, 2000 ; Ge et al, 2014 ). The short side chain of Ser makes it difficult for the OH groups of NAD-adenine moiety to interact with this residue in the coenzyme binding site (Ge et al, 2014 ).…”

Section: Attempts To Change the Coenzyme Specificity In Oxidoreductasmentioning

confidence: 99%

“…Ser usually interacts with the phosphate group of NADP stabilizing the coenzyme binding (Schepens et al, 2000 ; Ge et al, 2014 ). The short side chain of Ser makes it difficult for the OH groups of NAD-adenine moiety to interact with this residue in the coenzyme binding site (Ge et al, 2014 ). Therefore, in several studies a serine has been replaced to switch from NADP to NAD usage (Medina et al, 2001 ; Khoury et al, 2009 ), by Asp (Bastian et al, 2011 ; Brinkmann-Chen et al, 2013 ) and Arg (Chen et al, 1995 ; Rodríguez-Arnedo et al, 2005 ) respectively.…”

Section: Attempts To Change the Coenzyme Specificity In Oxidoreductasmentioning

confidence: 99%

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Protein Engineering for Nicotinamide Coenzyme Specificity in Oxidoreductases: Attempts and Challenges

Chánique

Parra

2018

Front. Microbiol.

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Oxidoreductases are ubiquitous enzymes that catalyze an extensive range of chemical reactions with great specificity, efficiency, and selectivity. Most oxidoreductases are nicotinamide cofactor-dependent enzymes with a strong preference for NADP or NAD. Because these coenzymes differ in stability, bioavailability and costs, the enzyme preference for a specific coenzyme is an important issue for practical applications. Different approaches for the manipulation of coenzyme specificity have been reported, with different degrees of success. Here we present various attempts for the switching of nicotinamide coenzyme preference in oxidoreductases by protein engineering. This review covers 103 enzyme engineering studies from 82 articles and evaluates the accomplishments in terms of coenzyme specificity and catalytic efficiency compared to wild type enzymes of different classes. We analyzed different protein engineering strategies and related them with the degree of success in inverting the cofactor specificity. In general, catalytic activity is compromised when coenzyme specificity is reversed, however when switching from NAD to NADP, better results are obtained. In most of the cases, rational strategies were used, predominantly with loop exchange generating the best results. In general, the tendency of removing acidic residues and incorporating basic residues is the strategy of choice when trying to change specificity from NAD to NADP, and vice versa. Computational strategies and algorithms are also covered as helpful tools to guide protein engineering strategies. This mini review aims to give a general introduction to the topic, giving an overview of tools and information to work in protein engineering for the reversal of coenzyme specificity.

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Section: Attempts To Change the Coenzyme Specificity In Oxidoreductasmentioning

confidence: 99%

Section: Attempts To Change the Coenzyme Specificity In Oxidoreductasmentioning

confidence: 99%

Protein Engineering for Nicotinamide Coenzyme Specificity in Oxidoreductases: Attempts and Challenges

Chánique

Parra

2018

Front. Microbiol.

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“…Interestingly, the genomes of M. alcaliphilum 20Z and three other gammaproteobacterial methanotrophs ( Methylomicrobium buryatense , Methylomicrobium album and Methylobacter tundripaludum ) possess sequences homologous to the LDH-like MDH (28% identities to M. trichosporium MDH) ( Figure S4 ). These sequences have unusual amino acids in the catalytic site at position 102 discriminating the substrate specificity of LDH and MDH enzymes: Thr ( M. alcaliphilum 20Z), Ser ( M. album ), Met ( M. buryatense ) and Asp ( M. tundripaludum ), instead of conservative Gln in LDH or Arg in MDH [ 18 , 31 ]. Determination of the products of these genes is in progress.…”

Section: Discussionmentioning

confidence: 99%

Role of NAD+-Dependent Malate Dehydrogenase in the Metabolism of Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b

et al. 2015

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We have expressed the l-malate dehydrogenase (MDH) genes from aerobic methanotrophs Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b as his-tagged proteins in Escherichia coli. The substrate specificities, enzymatic kinetics and oligomeric states of the MDHs have been characterized. Both MDHs were NAD+-specific and thermostable enzymes not affected by metal ions or various organic metabolites. The MDH from M. alcaliphilum 20Z was a homodimeric (2 × 35 kDa) enzyme displaying nearly equal reductive (malate formation) and oxidative (oxaloacetate formation) activities and higher affinity to malate (Km = 0.11 mM) than to oxaloacetate (Km = 0.34 mM). The MDH from M. trichosporium OB3b was homotetrameric (4 × 35 kDa), two-fold more active in the reaction of oxaloacetate reduction compared to malate oxidation and exhibiting higher affinity to oxaloacetate (Km = 0.059 mM) than to malate (Km = 1.28 mM). The kcat/Km ratios indicated that the enzyme from M. alcaliphilum 20Z had a remarkably high catalytic efficiency for malate oxidation, while the MDH of M. trichosporium OB3b was preferable for oxaloacetate reduction. The metabolic roles of the enzymes in the specific metabolism of the two methanotrophs are discussed.

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“…Malate dehydrogenase (MDH) of Streptomyces coelicolor ( Sc MDH) was altered by site-directed mutations in the Rossman fold region to change its cofactor specificity from NADH to NADPH. The coenzyme specificity ( K cat / K m ) of the mutant enzyme was examined and found to be shifted 2231.3-fold toward NADPH (Ge et al, 2014 ). Similarly, in Thermus thermophilus, Thermus flavus , and Thermococcus kodakarensis replacement of specific amino acids in Rossman fold of NADH dependent lactate dehydrogenase changed its cofactor from NADH to NADPH (Nishiyama et al, 1993 ; Tomita et al, 2006a ; Morimoto et al, 2014 ).…”

Section: Significance Of In Vitro Manipulationmentioning

confidence: 99%

In vitro Engineering of Novel Bioactivity in the Natural Enzymes

Tiwari

2016

Front. Chem.

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Enzymes catalyze various biochemical functions with high efficiency and specificity. In vitro design of the enzyme leads to novel bioactivity in this natural biomolecule that give answers of some vital questions like crucial residues in binding with substrate, molecular evolution, cofactor specificity etc. Enzyme engineering technology involves directed evolution, rational designing, semi-rational designing, and structure-based designing using chemical modifications. Similarly, combined computational and in vitro evolution approaches together help in artificial designing of novel bioactivity in the natural enzyme. DNA shuffling, error prone PCR and staggered extension process are used to artificially redesign active site of enzyme, which can alter its efficiency and specificity. Modifications of the enzyme can lead to the discovery of new path of molecular evolution, designing of efficient enzymes, locating active sites and crucial residues, shift in substrate, and cofactor specificity. The methods and thermodynamics of in vitro designing of the enzyme are also discussed. Similarly, engineered thermophilic and psychrophilic enzymes attain substrate specificity and activity of mesophilic enzymes that may also be beneficial for industry and therapeutics.

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Alteration of coenzyme specificity of malate dehydrogenase from Streptomyces coelicolor A3(2) by site-directed mutagenesis

Cited by 6 publications

References 23 publications

Protein Engineering for Nicotinamide Coenzyme Specificity in Oxidoreductases: Attempts and Challenges

Protein Engineering for Nicotinamide Coenzyme Specificity in Oxidoreductases: Attempts and Challenges

Role of NAD+-Dependent Malate Dehydrogenase in the Metabolism of Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b

In vitro Engineering of Novel Bioactivity in the Natural Enzymes

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