Crystal Structure and Inhibition Studies of Transglutaminase from Streptomyces mobaraense

Yang, Ming-Te; Chang, C.; Wang, Jiou Ming; Wu, Tung‐Kung; Wang, Yu Kuo; Chang, Chin-Yuan; Li, Tien Hsiung Thomas

doi:10.1074/jbc.m110.203315

Cited by 51 publications

(64 citation statements)

References 21 publications

(19 reference statements)

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“…(A) mTGase precursor structural overview. Surface representation of the active site cleft of mTGase shown in gray and cartoon representation of pro‐domain colored orange (PDB 3IU0) . Pro‐domain amino acid residues facing towards mTGase are shown in blue stick representation.…”

Section: Resultsmentioning

confidence: 99%

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Production of soluble and active microbial transglutaminase inEscherichia colifor site-specific antibody drug conjugation

et al. 2015

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Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro-domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine-scan of the mTGase pro-domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro-domain/ mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 208C, but results in full activity when shifted to 378C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro-domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30-75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site-specific antibody drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.

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Section: Resultsmentioning

confidence: 99%

“…Within the first short α‐helix residues Tyr10 and Tyr14 form the majority of the interactions with the enzyme active site [Fig. (B)] . Only one hydrophobic residue, Leu16, forms a short connective loop between α‐helices 1 and 2 of the mTGase pro‐domain and points deep into the active‐site cleft [Fig.…”

Section: Resultsmentioning

confidence: 99%

Production of soluble and active microbial transglutaminase inEscherichia colifor site-specific antibody drug conjugation

et al. 2015

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“…TGase is widely distributed in various organisms, including plants [3], mammals [4], and microorganisms [5]. Among the TGases, the TGase from Streptomyces is Ca 2+ -independent and is advantageous for industrial applications because it has a higher reaction rate, broad substrate specificity for an acyl donor, and a smaller molecular size [6, 7]. The development of an efficient and easy-to-use expression system for the production of Streptomyces TGase is therefore highly desirable.…”

Section: Introductionmentioning

confidence: 99%

Improving the active expression of transglutaminase in Streptomyces lividans by promoter engineering and codon optimization

Liu

Miao

et al. 2016

BMC Biotechnol

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BackgroundTransglutaminases (TGase), which are synthesized as a zymogen (pro-TGase) in Streptomyces sp., are important enzymes in the food industry. Because this pro-peptide is essential for the correct folding of Streptomyces TGase, TGase is usually expressed in an inactive pro-TGase form, which is then converted to active TGase by the addition of activating proteases in vitro. In this study, Streptomyces hygroscopicus TGase was actively produced by Streptomyces lividans through promoter engineering and codon optimization.ResultsA gene fragment (tg1, 2.6 kb) that encoded the pro-TGase and its endogenous promoter region, signal peptide and terminator was amplified from S. hygroscopicus WSH03-13 and cloned into plasmid pIJ86, which resulted in pIJ86/tg1. After fermentation for 2 days, S. lividans TK24 that harbored pIJ86/tg1 produced 1.8 U/mL of TGase, and a clear TGase band (38 kDa) was detected in the culture supernatant. These results indicated that the pro-TGase was successfully expressed and correctly processed into active TGase in S. lividans TK24 by using the TGase promoter. Based on deletion analysis, the complete sequence of the TGase promoter is restricted to the region from −693 to −48. We also identified a negative element (−198 to −148) in the TGase promoter, and the deletion of this element increased the TGase production by 81.3 %, in contrast to the method by which S. lividans expresses pIJ86/tg1. Combining the deletion of the negative element of the promoter and optimization of the gene codons, the yield and productivity of TGase reached 5.73 U/mL and 0.14 U/mL/h in the recombinant S. lividans, respectively.ConclusionsWe constructed an active TGase-producing strain that had a high yield and productivity, and the optimized TGase promoter could be a good candidate promoter for the expression of other proteins in Streptomyces.

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“…A negative aspect of farnesyl transferase labeling is that significant modifications of the farnesyl-pyrophosphate substrate reduce kinetic parameters by factors of 10–35 fold. 34 Transglutaminase offers perhaps the highest potential kinetics of irreversible peptide conjugation systems with k cat values approximately two orders of magnitude higher than Sfp with YbbR, 11,37 but the least site-specificity due to reaction with many possible glutamine-containing sequences. 35 Thus, for bioconjugation applications requiring short fusion sequences, easily reversible and site-specific labeling can be implemented with variations of the 11 amino acid YbbR using combined Sfp/PfAcpH methodology.…”

Section: Resultsmentioning

confidence: 99%

Chemoenzymatic exchange of phosphopantetheine on protein and peptide

2014

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Evaluation of new acyl carrier protein hydrolase (AcpH, EC 3.1.4.14) homologs from proteobacteria and cyanobacteria reveals significant variation in substrate selectivity and kinetic parameters for phosphopantetheine hydrolysis from carrier proteins. Evaluation with carrier proteins from both primary and secondary metabolic pathways reveals an overall preference for acyl carrier protein (ACP) substrates from type II fatty acid synthases, as well as variable activity for polyketide synthase ACPs and peptidyl carrier proteins (PCP) from non-ribosomal peptide synthases. We also demonstrate the kinetic parameters of these homologs for AcpP and the 11-mer peptide substrate YbbR. These findings enable the fully reversible labeling of all three classes of natural product synthase carrier proteins as well as full and minimal fusion protein constructs.

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Crystal Structure and Inhibition Studies of Transglutaminase from Streptomyces mobaraense

Cited by 51 publications

References 21 publications

Production of soluble and active microbial transglutaminase inEscherichia colifor site-specific antibody drug conjugation

Production of soluble and active microbial transglutaminase inEscherichia colifor site-specific antibody drug conjugation

Improving the active expression of transglutaminase in Streptomyces lividans by promoter engineering and codon optimization

Chemoenzymatic exchange of phosphopantetheine on protein and peptide

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