scholarly journals Identification of the catalytic residues of the first family of β(1–3)glucanosyltransferases identified in fungi

2000 ◽  
Vol 347 (3) ◽  
pp. 741-747 ◽  
Author(s):  
Isabelle MOUYNA ◽  
Michel MONOD ◽  
Thierry FONTAINE ◽  
Bernard HENRISSAT ◽  
Barbara LÉCHENNE ◽  
...  
Keyword(s):  
Cluster Analysis ◽  
Active Site ◽  
Biochemical Analysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Sequence Comparisons ◽  
Catalytic Residues ◽  
Hydrophobic Cluster Analysis ◽  
Hydrophobic Cluster ◽  
New Family

A new family of glycosylphosphatidylinositol-anchored β(1-3)glucanosyltransferases (Gelp), recently identified and characterized in the filamentous fungus Aspergillus fumigatus, showed functional similarity to the Gas/Phr/Epd protein families, which are involved in yeast morphogenesis. Sequence comparisons and hydrophobic cluster analysis (HCA) showed that all the Gas/Phr/Epd/Gel proteins belong to a new family of glycosylhydrolases, family 72. We confirmed by site-directed mutagenesis and biochemical analysis that the two conserved glutamate residues (the putative catalytic residues of this family, as determined by HCA) are involved in the active site of this family of glycosylhydrolases.

scholarly journals Evidence that Asn542 of neprilysin (EC 3.4.24.11) is involved in binding of the P2′ residue of substrates and inhibitors

1995 ◽  
Vol 311 (2) ◽  
pp. 623-627 ◽  
Author(s):  
N Dion ◽  
H Le Moual ◽  
M C Fournié-Zaluski ◽  
B P Roques ◽  
P Crine ◽  
...  
Keyword(s):  
Active Site ◽  
Substrate Binding ◽  
Biologically Active ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Hydrophobic Cluster Analysis ◽  
Active Peptides ◽  
Ic50 Values

Neprilysin (EC 3.4.24.11) is a Zn2+ metallopeptidase involved in the degradation of biologically active peptides, e.g. enkephalins and atrial natriuretic peptide. The substrate specificity and catalytic activity of neprilysin resemble those of thermolysin, a crystallized bacterial Zn2+ metalloprotease. Despite little overall homology between the primary structures of thermolysin and neprilysin, many of the amino acid residues involved in catalysis, as well as Zn2+ and substrate binding, are highly conserved. Most of the active-site residues of neprilysin have their homologues in thermolysin and have been characterized by site-directed mutagenesis. Furthermore, hydrophobic cluster analysis has revealed some other analogies between the neprilysin and thermolysin sequences [Benchetrit, Bissery, Mornon, Devault, Crine and Roques (1988) Biochemistry 27, 592-596]. According to this analysis the role of Asn542 in the neprilysin active site is analogous to that of Asn112 of thermolysin, which is to bind the substrate. Site-directed mutagenesis was used to change Asn542 to Gly or Gln residues. The effect of these mutations on substrate catalysis and inhibitor binding was examined with a series of thiorphan-like compounds containing various degrees of methylation at the P2′ residue. For both mutated enzymes, determination of kinetic parameters with [D-Ala2,Leu5]enkephalin as substrate showed that the large decrease in activity was attributable to an increase in Km (14-16-fold) whereas kcat values were only slightly affected (2-3-fold decrease). This is in agreement with Asn542 being involved in substrate binding rather than directly in catalysis. Finally, the IC50 values for thiorphan and substituted thiorphans strongly suggest that Asn542 of neprilysin binds the substrate on the amino side of the P2′ residue by formation of a unique hydrogen bond.

A new group of plant-specific ATP-dependent DNA ligases identified by protein phylogeny, hydrophobic cluster analysis and 3-dimensional modelling

2005 ◽  
Vol 32 (2) ◽  
pp. 161 ◽  
Author(s):  
Diego Bonatto ◽  
Martin Brendel ◽  
João Antonio Pêgas Henriques
Keyword(s):  
Cluster Analysis ◽  
Oryza Sativa L ◽  
Chemical Properties ◽  
Animal Cells ◽  
Hydrophobic Cluster Analysis ◽  
Dna Ligases ◽  
3 Dimensional ◽  
Hydrophobic Cluster ◽  
New Family ◽  
Brassica Spp

The eukaryotic ATP-dependent DNA ligases comprise a group of orthologous proteins that have distinct roles in DNA metabolism. In contrast with the well-known DNA ligases of animal cells, the DNA ligases of plant cells are poorly described. Until now, only two DNA ligases (I and IV) genes of Arabidopsis thaliana (L.) Heynh were isolated and characterised. Use of the complete genomic sequences of Oryza sativa L. and A. thaliana, as well as the partially assembled genomic data of Medicago truncatula L. and Brassica spp., allowed us to identify a new family of ATP-dependent DNA ligases that are found only in the Viridiplantae kingdom. An in-depth phylogenetic analysis of protein sequences showed that this family composes a distinct clade, which shares a last universal common ancestor with DNA ligases I. In silico sequence studies indicate that these proteins have distinct physico-chemical properties when compared with those of animal and fungal DNA ligases. Moreover, hydrophobic cluster analysis and 3-dimensional modelling allowed us to map two conserved domains within these DNA ligases I-like proteins. Additional data of microsynteny analysis indicate that these DNA ligases I-like genes are linked to the S and SLL2 loci of Brassica spp. and A. thaliana, respectively. Combining the results of all analyses, we propose the creation of the DNA ligases VI (LIG6) family, which is composed by plant-specific DNA ligases.

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 Active-site motifs of lysosomal acid hydrolases: invariant features of clan GH-A glycosyl hydrolases deduced from hydrophobic cluster analysis

Glycobiology ◽  
1997 ◽  
Vol 7 (2) ◽  
pp. 277-284 ◽  
Author(s):  
Patrick Durand ◽  
Pierre Lehn ◽  
Isabelle Callebaut ◽  
Sylvie Fabrega ◽  
Bernard Henrissat ◽  
...  
Keyword(s):  
Cluster Analysis ◽  
Active Site ◽  
Glycosyl Hydrolases ◽  
Acid Hydrolases ◽  
Lysosomal Acid ◽  
Hydrophobic Cluster Analysis ◽  
Hydrophobic Cluster ◽  
Invariant Features

scholarly journals Structural Insights into l-Tryptophan Dehydrogenase from a Photoautotrophic Cyanobacterium, Nostoc punctiforme

2016 ◽  
Vol 83 (2) ◽  
Author(s):  
Taisuke Wakamatsu ◽  
Haruhiko Sakuraba ◽  
Megumi Kitamura ◽  
Yuichi Hakumai ◽  
Kenji Fukui ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Folding ◽  
Amino Acid ◽  
Active Site ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Nostoc Punctiforme ◽  
Hydrophobic Cluster ◽  
High Amino Acid

ABSTRACT l-Tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH), despite exhibiting high amino acid sequence identity (>30%)/homology (>50%) with NAD(P)+-dependent l-Glu/l-Leu/l-Phe/l-Val dehydrogenases, exclusively catalyzes reversible oxidative deamination of l-Trp to 3-indolepyruvate in the presence of NAD+. Here, we determined the crystal structure of the apo form of NpTrpDH. The structure of the NpTrpDH monomer, which exhibited high similarity to that of l-Glu/l-Leu/l-Phe dehydrogenases, consisted of a substrate-binding domain (domain I, residues 3 to 133 and 328 to 343) and an NAD+/NADH-binding domain (domain II, residues 142 to 327) separated by a deep cleft. The apo-NpTrpDH existed in an open conformation, where domains I and II were apart from each other. The subunits dimerized themselves mainly through interactions between amino acid residues around the β-1 strand of each subunit, as was observed in the case of l-Phe dehydrogenase. The binding site for the substrate l-Trp was predicted by a molecular docking simulation and validated by site-directed mutagenesis. Several hydrophobic residues, which were located in the active site of NpTrpDH and possibly interacted with the side chain of the substrate l-Trp, were arranged similarly to that found in l-Leu/l-Phe dehydrogenases but fairly different from that of an l-Glu dehydrogenase. Our crystal structure revealed that Met-40, Ala-69, Ile-74, Ile-110, Leu-288, Ile-289, and Tyr-292 formed a hydrophobic cluster around the active site. The results of the site-directed mutagenesis experiments suggested that the hydrophobic cluster plays critical roles in protein folding, l-Trp recognition, and catalysis. Our results provide critical information for further characterization and engineering of this enzyme. IMPORTANCE In this study, we determined the three-dimensional structure of l-Trp dehydrogenase, analyzed its various site-directed substitution mutants at residues located in the active site, and obtained the following informative results. Several residues in the active site form a hydrophobic cluster, which may be a part of the hydrophobic core essential for protein folding. To our knowledge, there is no previous report demonstrating that a hydrophobic cluster in the active site of any l-amino acid dehydrogenase may have a critical impact on protein folding. Furthermore, our results suggest that this hydrophobic cluster could strictly accommodate l-Trp. These studies show the structural characteristics of l-Trp dehydrogenase and hence would facilitate novel applications of l-Trp dehydrogenase.

Sign in / Sign up

Export Citation Format

Share Document