Discovery of a microbial transglutaminase enabling highly site-specific labeling of proteins

Steffen, Wojtek; Ko, Fu Chong; Patel, Jigar; Lyamichev, Victor I.; Albert, Thomas; Benz, Jörg; Rudolph, M.G.; Bergmann, Frank; Streidl, Thomas; Kratzsch, Peter; Boenitz-Dulat, Mara; Oelschlaegel, Tobias; Schraeml, Michael

doi:10.1074/jbc.m117.797811

Cited by 51 publications

(67 citation statements)

References 51 publications

(56 reference statements)

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“…It appears that almost all treatments have the same pattern of molecular weight distribution, between 60 kDa and 190 kDa. This result is higher than that reported by previous study (Wulandari et al, 2016) that gelatin from bovine split hide has a range of molecular weight of 25-40 kDa, and higher than the molecular weight of transglutaminase enzyme from Streptomyces or Bacilli (≥38 kDa) (Steffen et al, 2017). Kyriakopoulou et al (2019) stated that SPI was one of the proteins containing cystine and cysteine that formed sulfide bonds.…”

Section: Discussionmentioning

confidence: 53%

Improvement of Bovine Split Hide Gelatin Quality by Addition of Soy Protein Isolate Using Transglutaminase Enzyme

Wulandari

Erwanto

Pranoto

et al. 2019

Trop. Anim. Sci. J.

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Bovine split hide is the subcutis layer with the low percentage of collagen so that the quality of the gelatin is different from that of the gelatin from cattle's skin. This study aims to improve the characteristics of bovine split hide gelatin combined with soy protein isolate (SPI) using transglutaminase (TGase) enzyme as a protein cross-linking agent. The study was conducted using a completely randomized design with 3 x 3 factorial pattern consisting of three levels of a mixture of bovine split hide gelatin : SPI at the ratios of 90 : 10, 80 : 20, 70 : 30, and three concentrations of transglutaminase enzyme, i.e. 10, 20, and 30 U. The results showed that treatments significantly affected viscosity, gel strength, the moisture, ash, fat, and protein content of gelatin. Electrophoresis of gelatin protein showed bands distribution between 60-190 kDa. The amino acid profile of the gelatin was similar to that of collagen with a high level of aspartate, glutamate, cysteine, and proline. Morphology of gelatin was observed by Scanning Electronic Microscope (SEM) and showed a compact distributed collagen crosslink. The combination of gelatin bovine split hide and SPI at the ratio of 90 : 10 provides the best physicochemical characteristic.

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Section: Discussionmentioning

confidence: 53%

Improvement of Bovine Split Hide Gelatin Quality by Addition of Soy Protein Isolate Using Transglutaminase Enzyme

Wulandari

Erwanto

Pranoto

et al. 2019

Trop. Anim. Sci. J.

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“…Type (H)XC 3 Adenain 4WX4_A 1.03 N/H54-OG1/T45 3.5 O/H54-N/T45 2.9 N/M56-O/V43 2.9 O/M56-N/V43 2.8 N/A57-O/F70 2.9 O/A57-N/F70 2.8 OD1/D71-NE2/H54 2.8 N/W55-SG/C122 3.5 OD1/D71-N/F73 2.9 Cys122 Asp71 [ 36 ] IV. Type C( X )H 4 Microbial transglutaminase 5M6Q_A 1.98 N/D175-O/R131 3.0 O/D175-N/R131 2.9 N/G177-O/S129 2.8 O/G177-N/S129 2.8 N/W178-O/W187 2.8 O/W178-N/W187 2.9 ND1/H188-OD2/D175 3.1 N/Y176-SG/C46 3.6 Cys46 His188 [ 37 ] V. Type CXH 5 Cysteine protease ATG4B 2CY7_A 1.90 N/S262-O/K259 2.8 O/S262-HOH425 2.6 HOH425-N/K259 2.9 O/H264-N/G257 3.2 N/H264-O/G257 2.9 N/Y265-O/L277 3.2 O/Y265-N/L277 2.9 OD1/D278-ND1/H280 2.6 N/A263-SG/C74 3.6 OD1/D278-N/H280 2.6 Cys74 Asp278 [ 38 ] PCP-Zone: Class B PCP-SCC: VI. Type XC(H) 6 Papain-like protease 2 4IUM_A 1.45 N/H332-O/D329 3.0 O/H332-N/D329 2.9 N/R334-O/I327 2.9 O/R334-N/I327 2.8 N/V335-O/Y262 2.8 O/V335-N/Y262 2.9 OD1/N263-NE2/H332 2.9 N/W333-SG/C270 3.7 Cys270 Asn263 [ 39 ] VII.…”

Section: Resultsmentioning

confidence: 99%

Papain-like cysteine proteinase zone (PCP-zone) and PCP structural catalytic core (PCP-SCC) of enzymes with cysteine proteinase fold

Denessiouk

Uversky

Permyakov

et al. 2020

International Journal of Biological Macromolecules

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There are several families of cysteine proteinases with different folds – for example the (chymo)trypsin fold family and papain-like fold family – but in both families the hydrolase activity of cysteine proteinases requires a cysteine residue as the catalytic nucleophile. In this work, we have analyzed the topology of the active site regions in 146 three-dimensional structures of proteins belonging to the Papain-like Cysteine Proteinase (PCP) superfamily, which includes papain as a typical representative of this protein superfamily. All analyzed enzymes contain a unique structurally closed conformation – a “PCP-Zone” – which can be divided into two groups, Class A and Class B. Eight structurally conserved amino acids of the PCP-Zone form a common Structural Core. The Structural Core, catalytic nucleophile, catalytic base and residue Xaa – which stabilizes the side-chain conformation of the catalytic base – make up a PCP Structural Catalytic Core (PCP-SCC). The PCP-SCC of Class A and Class B are divided into 5 and 2 types, respectively. Seven variants of the mutual arrangement of the amino-acid side chains of the catalytic triad – nucleophile, base and residue Xaa – within the same fold clearly demonstrate how enzymes with the papain-like fold adapt to the need to perform diverse functions in spite of their limited structural diversity. The roles of both the PCP-Zone of SARS-CoV-2-PLpro described in this study and the NBCZone of SARS-CoV-2-3CLpro presented in our earlier article (Denesyuk AI, Johnson MS, Salo-Ahen OMH, Uversky VN, Denessiouk K. Int J Biol Macromol. 2020;153:399-411) that are in contacts with inhibitors are discussed.

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“…Since we performed these experiments, another TGase has been reported [ 41 ], allowing us another point of comparison. The microbial TGase from Kutzneria albida (KalbTG) was expressed as a soluble 26-kDa enzyme, and a highly specific glutamine substrate sequence, namely GGGYRYRQGGGG, was engineered for the bioconjugation application of KalbTG.…”

Section: Resultsmentioning

confidence: 99%

Design of a glutamine substrate tag enabling protein labelling mediated by Bacillus subtilis transglutaminase

2018

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Transglutaminases (TGases) are enzymes that catalyse protein cross-linking through a transamidation reaction between the side chain of a glutamine residue on one protein and the side chain of a lysine residue on another. Generally, TGases show low substrate specificity with respect to their amine substrate, such that a wide variety of primary amines can participate in the modification of specific glutamine residue. Although a number of different TGases have been used to mediate these bioconjugation reactions, the TGase from Bacillus subtilis (bTG) may be particularly suited to this application. It is smaller than most TGases, can be expressed in a soluble active form, and lacks the calcium dependence of its mammalian counterparts. However, little is known regarding this enzyme and its glutamine substrate specificity, limiting the scope of its application. In this work, we designed a FRET-based ligation assay to monitor the bTG-mediated conjugation of the fluorescent proteins Clover and mRuby2. This assay allowed us to screen a library of random heptapeptide glutamine sequences for their reactivity with recombinant bTG in bacterial cells, using fluorescence assisted cell sorting. From this library, several reactive sequences were identified and kinetically characterized, with the most reactive sequence (YAHQAHY) having a kcat/KM value of 19 ± 3 μM-1 min-1. This sequence was then genetically appended onto a test protein as a reactive ‘Q-tag’ and fluorescently labelled with dansyl-cadaverine, in the first demonstration of protein labelling mediated by bTG.

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Discovery of a microbial transglutaminase enabling highly site-specific labeling of proteins

Cited by 51 publications

References 51 publications

Improvement of Bovine Split Hide Gelatin Quality by Addition of Soy Protein Isolate Using Transglutaminase Enzyme

Improvement of Bovine Split Hide Gelatin Quality by Addition of Soy Protein Isolate Using Transglutaminase Enzyme

Papain-like cysteine proteinase zone (PCP-zone) and PCP structural catalytic core (PCP-SCC) of enzymes with cysteine proteinase fold

Design of a glutamine substrate tag enabling protein labelling mediated by Bacillus subtilis transglutaminase

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