scholarly journals 1.68-Å Crystal Structure of Endopolygalacturonase II fromAspergillus nigerand Identification of Active Site Residues by Site-directed Mutagenesis

1999 ◽  
Vol 274 (43) ◽  
pp. 30474-30480 ◽  
Author(s):  
Yovka van Santen ◽  
Jacques A. E. Benen ◽  
Klaus-Hasso Schröter ◽  
Kor H. Kalk ◽  
Sylvie Armand ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Active Site Residues

scholarly journals Crystal Structure and Desulfurization Mechanism of 2′-Hydroxybiphenyl-2-sulfinic Acid Desulfinase

2006 ◽  
Vol 281 (43) ◽  
pp. 32534-32539 ◽  
Author(s):  
Woo Cheol Lee ◽  
Takashi Ohshiro ◽  
Toshiyuki Matsubara ◽  
Yoshikazu Izumi ◽  
Masaru Tanokura
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Structural Changes ◽  
Hydrophobic Interactions ◽  
Rhodococcus Erythropolis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
Sulfinic Acid ◽  
New Family

The desulfurization of dibenzothiophene in Rhodococcus erythropolis is catalyzed by two monooxygenases, DszA and DszC, and a desulfinase, DszB. In the last step of this pathway, DszB hydrolyzes 2′-hydroxybiphenyl-2-sulfinic acid into 2-hydroxybiphenyl and sulfite. We report on the crystal structures of DszB and an inactive mutant of DszB in complex with substrates at resolutions of 1.8Å or better. The overall fold of DszB is similar to those of periplasmic substrate-binding proteins. In the substrate complexes, biphenyl rings of substrates are recognized by extensive hydrophobic interactions with the active site residues. Binding of substrates accompanies structural changes of the active site loops and recruits His60 to the active site. The sulfinate group of bound substrates forms hydrogen bonds with side chains of Ser27, His60, and Arg70, each of which is shown by site-directed mutagenesis to be essential for the activity. In our proposed reaction mechanism, Cys27 functions as a nucleophile and seems to be activated by the sulfinate group of substrates, whereas His60 and Arg70 orient the syn orbital of sulfinate oxygen to the sulfhydryl hydrogen of Cys27 and stabilize the negatively charged reaction intermediate. Cys, His, and Arg residues are conserved in putative proteins homologous to DszB, which are presumed to constitute a new family of desulfinases.

scholarly journals Threonine 57 is required for the post-translational activation ofEscherichia coliaspartate α-decarboxylase

2014 ◽  
Vol 70 (4) ◽  
pp. 1166-1172 ◽  
Author(s):  
Michael E. Webb ◽  
Briony A. Yorke ◽  
Tom Kershaw ◽  
Sarah Lovelock ◽  
Carina M. C. Lobley ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Intramolecular Rearrangement ◽  
Directed Mutagenesis ◽  
Vitamin B ◽  
Biosynthesis Pathway ◽  
Serine Residue ◽  
Translational Activation

Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formedviathe intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.

scholarly journals Site-directed mutagenesis of active site residues in a class I endochitinase from chestnut seeds

Glycobiology ◽  
1998 ◽  
Vol 8 (10) ◽  
pp. 1021-1028 ◽  
Author(s):  
G. Garcia-Casado ◽  
C. Collada ◽  
I. Allona ◽  
R. Casado ◽  
L. F. Pacios ◽  
...  
Keyword(s):  
Active Site ◽  
Site Directed Mutagenesis ◽  
Class I ◽  
Directed Mutagenesis ◽  
Active Site Residues

scholarly journals Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli

1994 ◽  
Vol 303 (2) ◽  
pp. 357-362 ◽  
Author(s):  
M P G van der Linden ◽  
L de Haan ◽  
O Dideberg ◽  
W Keck
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Binding Capacity ◽  
Binding Protein ◽  
Complete Loss ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Active Site Residues

Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis.

scholarly journals Identification of core active site residues of the sulfur oxygenase reductase fromAcidianus ambivalensby site-directed mutagenesis

2005 ◽  
Vol 248 (2) ◽  
pp. 171-176 ◽  
Author(s):  
Tim Urich ◽  
Anja Kroke ◽  
Christian Bauer ◽  
Kerstin Seyfarth ◽  
Muriel Reuff ◽  
...  
Keyword(s):  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
Sulfur Oxygenase Reductase
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