Investigation of Structural Determinants for the Substrate Specificity in the Zinc-Dependent Alcohol Dehydrogenase CPCR2 fromCandida parapsilosis

ChemBioChem ◽  
2015 ◽  
Vol 16 (10) ◽  
pp. 1512-1519 ◽  
Author(s):  
Christoph Loderer ◽  
Gaurao V. Dhoke ◽  
Mehdi D. Davari ◽  
Wolfgang Kroutil ◽  
Ulrich Schwaneberg ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Substrate Specificity ◽  
Structural Determinants

scholarly journals Human and mouse granzyme M display divergent and species-specific substrate specificities

2011 ◽  
Vol 437 (3) ◽  
pp. 431-442 ◽  
Author(s):  
Stefanie A.H. de Poot ◽  
Marijn Westgeest ◽  
Daniel R. Hostetter ◽  
Petra van Damme ◽  
Kim Plasman ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Specific Substrate ◽  
Structural Determinants ◽  
Cytotoxic Lymphocyte ◽  
Potent Inducer ◽  
Methionine Residue ◽  
Positional Scanning ◽  
Methionine Residues ◽  
Species Specific ◽  
Positional Scanning Libraries

Cytotoxic lymphocyte protease GrM (granzyme M) is a potent inducer of tumour cell death and a key regulator of inflammation. Although hGrM (human GrM) and mGrM (mouse GrM) display extensive sequence homology, the substrate specificity of mGrM remains unknown. In the present study, we show that hGrM and mGrM have diverged during evolution. Positional scanning libraries of tetrapeptide substrates revealed that mGrM is preferred to cleave after a methionine residue, whereas hGrM clearly favours a leucine residue at the P1 position. The kinetic optimal non-prime subsites of both granzymes were also distinct. Gel-based and complementary positional proteomics showed that hGrM and mGrM have a partially overlapping set of natural substrates and a diverged prime and non-prime consensus cleavage motif with leucine and methionine residues being major P1 determinants. Consistent with positional scanning libraries of tetrapeptide substrates, P1 methionine was more frequently used by mGrM as compared with hGrM. Both hGrM and mGrM cleaved α-tubulin with similar kinetics. Strikingly, neither hGrM nor mGrM hydrolysed mouse NPM (nucleophosmin), whereas human NPM was hydrolysed efficiently by GrM from both species. Replacement of the putative P1′–P2′ residues in mouse NPM with the corresponding residues of human NPM restored cleavage of mouse NPM by both granzymes. This further demonstrates the importance of prime sites as structural determinants for GrM substrate specificity. GrM from both species efficiently triggered apoptosis in human but not in mouse tumour cells. These results indicate that hGrM and mGrM not only exhibit divergent specificities but also trigger species-specific functions.

Structural insights into substrate specificity and solvent tolerance in alcohol dehydrogenase ADH-‘A’ from Rhodococcus ruber DSM 44541

2010 ◽  
Vol 46 (34) ◽  
pp. 6314 ◽  
Author(s):  
Martin Karabec ◽  
Andrzej Łyskowski ◽  
Katharina C. Tauber ◽  
Georg Steinkellner ◽  
Wolfgang Kroutil ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Substrate Specificity ◽  
Solvent Tolerance ◽  
Rhodococcus Ruber ◽  
Structural Insights

Substrate specificity of alcohol dehydrogenase from the yeastHansenyls polymorpha CBS 4732 andCandida utilis CBS 621

Yeast ◽  
1988 ◽  
Vol 4 (2) ◽  
pp. 143-148 ◽  
Author(s):  
Cornelis Verduyn ◽  
Guido J. Breedveld ◽  
W. Alexander Scheffers ◽  
Johnnes P. Van Dijken
Keyword(s):  
Alcohol Dehydrogenase ◽  
Substrate Specificity

scholarly journals Characterization of Hybrid Toluate and Benzoate Dioxygenases

2003 ◽  
Vol 185 (18) ◽  
pp. 5333-5341 ◽  
Author(s):  
Yong Ge ◽  
Lindsay D. Eltis
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Acinetobacter Calcoaceticus ◽  
The Other ◽  
Β Subunit ◽  
Structural Determinants ◽  
Α Subunit ◽  
Benzoate Dioxygenase

ABSTRACT Toluate dioxygenase of Pseudomonas putida mt-2 (TADOmt2) and benzoate dioxygenase of Acinetobacter calcoaceticus ADP1 (BADOADP1) catalyze the 1,2-dihydroxylation of different ranges of benzoates. The catalytic component of these enzymes is an oxygenase consisting of two subunits. To investigate the structural determinants of substrate specificity in these ring-hydroxylating dioxygenases, hybrid oxygenases consisting of the α subunit of one enzyme and the β subunit of the other were prepared, and their respective specificities were compared to those of the parent enzymes. Reconstituted BADOADP1 utilized four of the seven tested benzoates in the following order of apparent specificity: benzoate > 3-methylbenzoate > 3-chlorobenzoate > 2-methylbenzoate. This is a significantly narrower apparent specificity than for TADOmt2 (3-methylbenzoate > benzoate ∼ 3-chlorobenzoate > 4-methylbenzoate ∼ 4-chlorobenzoate ≫ 2-methylbenzoate ∼ 2-chlorobenzoate [Y. Ge, F. H. Vaillancourt, N. Y. Agar, and L. D. Eltis, J. Bacteriol. 184:4096-4103, 2002]). The apparent substrate specificity of the αBβT hybrid oxygenase for these benzoates corresponded to that of BADOADP1, the parent from which the α subunit originated. In contrast, the apparent substrate specificity of the αTβB hybrid oxygenase differed slightly from that of TADOmt2 (3-chlorobenzoate > 3-methylbenzoate > benzoate ∼ 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chlorobenzoate). Moreover, the αTβB hybrid catalyzed the 1,6-dihydroxylation of 2-methylbenzoate, not the 1,2-dihydroxylation catalyzed by the TADOmt2 parent. Finally, the turnover of this ortho-substituted benzoate was much better coupled to O2 utilization in the hybrid than in the parent. Overall, these results support the notion that the α subunit harbors the principal determinants of specificity in ring-hydroxylating dioxygenases. However, they also demonstrate that the β subunit contributes significantly to the enzyme's function.

Role of Tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase

Extremophiles ◽  
2009 ◽  
Vol 13 (5) ◽  
pp. 751-761 ◽  
Author(s):  
Angela Pennacchio ◽  
Luciana Esposito ◽  
Adriana Zagari ◽  
Mosè Rossi ◽  
Carlo A. Raia
Keyword(s):  
Alcohol Dehydrogenase ◽  
Substrate Specificity ◽  
Structural Stability ◽  
Sulfolobus Solfataricus
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