Reactive cysteine in the active-site motif of Bacteroides thetaiotaomicron dipeptidyl peptidase III is a regulatory residue for enzyme activity

2012 ◽  
Vol 393 (1-2) ◽  
pp. 37-46 ◽  
Author(s):  
Bojana Vukelić ◽  
Branka Salopek-Sondi ◽  
Jasminka Špoljarić ◽  
Igor Sabljić ◽  
Nevenka Meštrović ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Sequence Similarity ◽  
Catalytic Efficiency ◽  
Basic Amino Acid ◽  
Dipeptidyl Peptidase ◽  
Pain Modulation ◽  
Binding Motif ◽  
Bacteroides Thetaiotaomicron ◽  
Active Site Motif

Abstract Dipeptidyl peptidase III (DPP III), a member of the metallopeptidase family M49, was considered as an exclusively eukaryotic enzyme involved in intracellular peptide cata­bolism and pain modulation. In 2003, new data on genome sequences revealed the first prokaryotic orthologs, which showed low sequence similarity to eukaryotic ones and a cysteine (Cys) residue in the zinc-binding motif HEXXGH. Here we report the cloning and heterologous expression of DPP III from the human gut symbiont Bacteroides thetaiotaomicron. The catalytic efficiency of bacterial DPP III for preferred synthetic substrate hydrolysis was very similar to that of the human host enzyme. Substitution of Cys450 from the active-site motif by serine did not substantially change the enzymatic activity. However, this residue was wholly responsible for the inactivation effect of sulfhydryl reagents. Molecular modeling indicated seven basic amino acid residues in the local environment of Cys450 as a possible cause for its high reactivity. Sequence analysis of 81 bacterial M49 peptidases showed conservation of the HECLGH motif in 68 primary structures with the majority of proteins lacking an active-site Cys originated from aerobic bacteria. Data obtained suggest that Cys450 of B. thetaiotaomicron DPP III is a regulatory residue for the enzyme activity.

scholarly journals Dynamic properties of dipeptidyl peptidase III from Bacteroides thetaiotaomicron and the structural basis for its substrate specificity – a computational study

2017 ◽  
Vol 13 (11) ◽  
pp. 2407-2417 ◽  
Author(s):  
M. Tomin ◽  
S. Tomić
Keyword(s):  
Enzyme Activity ◽  
Substrate Specificity ◽  
Computational Study ◽  
Dynamic Properties ◽  
Dipeptidyl Peptidase ◽  
Structural Basis ◽  
Wild Type ◽  
Bacteroides Thetaiotaomicron ◽  
Human Gut

Dynamics and enzyme activity of dipeptidyl peptidase III, wild type and mutants, from the human gut symbiont Bacteroides thetaiotaomicron.

scholarly journals A novel plant enzyme with dual activity: an atypical Nudix hydrolase and a dipeptidyl peptidase III

2017 ◽  
Vol 398 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Zrinka Karačić ◽  
Bojana Vukelić ◽  
Gabrielle H. Ho ◽  
Iva Jozić ◽  
Iva Sučec ◽  
...  
Keyword(s):  
Physcomitrella Patens ◽  
Catalytic Efficiency ◽  
Peptide Bond ◽  
Dipeptidyl Peptidase ◽  
Biochemical Properties ◽  
Binding Motif ◽  
Nudix Hydrolase ◽  
Purine Nucleotides ◽  
Uncharacterized Protein ◽  
Hydrolysis Of

Abstract In a search for plant homologues of dipeptidyl peptidase III (DPP III) family, we found a predicted protein from the moss Physcomitrella patens (UniProt entry: A9TLP4), which shared 61% sequence identity with the Arabidopsis thaliana uncharacterized protein, designated Nudix hydrolase 3. Both proteins contained all conserved regions of the DPP III family, but instead of the characteristic hexapeptide HEXXGH zinc-binding motif, they possessed a pentapeptide HEXXH, and at the N-terminus, a Nudix box, a hallmark of Nudix hydrolases, known to act upon a variety of nucleoside diphosphate derivatives. To investigate their biochemical properties, we expressed heterologously and purified Physcomitrella (PpND) and Arabidopsis (AtND) protein. Both hydrolyzed, with comparable catalytic efficiency, the isopentenyl diphosphate (IPP), a universal precursor for the biosynthesis of isoprenoid compounds. In addition, PpND dephosphorylated four purine nucleotides (ADP, dGDP, dGTP, and 8-oxo-dATP) with strong preference for oxidized dATP. Furthermore, PpND and AtND showed DPP III activity against dipeptidyl-2-arylamide substrates, which they cleaved with different specificity. This is the first report of a dual activity enzyme, highly conserved in land plants, which catalyzes the hydrolysis of a peptide bond and of a phosphate bond, acting both as a dipeptidyl peptidase III and an atypical Nudix hydrolase.

The metal-binding motif of dipeptidyl peptidase III directly influences the enzyme activity in the copper derivative of dipeptidyl peptidase III

2004 ◽  
Vol 431 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Junzo Hirose ◽  
Hiroshi Kamigakiuchi ◽  
Hiroyuki Iwamoto ◽  
Hideaki Fujii ◽  
Masanori Nakai ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Metal Binding ◽  
Dipeptidyl Peptidase ◽  
Binding Motif ◽  
Metal Binding Motif

scholarly journals A Novel β-N-Acetylglucosaminidase fromStreptomyces thermoviolaceus OPC-520: Gene Cloning, Expression, and Assignment to Family 3 of the Glycosyl Hydrolases

1998 ◽  
Vol 64 (8) ◽  
pp. 2920-2924 ◽  
Author(s):  
Hiroshi Tsujibo ◽  
Naoya Hatano ◽  
Tadahisa Mikami ◽  
Ayako Hirasawa ◽  
Katsushiro Miyamoto ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Sequence Similarity ◽  
Glycosyl Hydrolase ◽  
Open Reading Frame ◽  
Glycosyl Hydrolases ◽  
Terminal Amino Acid ◽  
Reading Frame ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

ABSTRACT A β-N-acetylglucosaminidase gene (nagA) of Streptomyces thermoviolaceus OPC-520 was cloned inStreptomyces lividans 66. The nucleotide sequence of the gene, which encodes NagA, revealed an open reading frame of 1,896 bp, encoding a protein with an M r of 66,329. The deduced primary structure of NagA was confirmed by comparison with the N-terminal amino acid sequence of the cloned β-N-acetylglucosaminidase expressed by S. lividans. The enzyme shares no sequence similarity with the classical β-N-acetylglucosaminidases belonging to family 20. However, NagA, which showed no detectable β-glucosidase activity, revealed homology with microbial β-glucosidases belonging to family 3; in particular, striking homology with the active-site regions of β-glucosidases was observed. Thus, the above-mentioned results indicate that NagA from S. thermoviolaceus OPC-520 is classified as a family 3 glycosyl hydrolase. The enzyme activity was optimal at 60°C and pH 5.0, and the apparentKm and V max values forp-nitrophenyl-β-N-acetylglucosamine were 425.7 μM and 24.8 μmol min−1 mg of protein−1, respectively.

scholarly journals New Insights into the Fructosyltransferase Activity of Schwanniomyces occidentalis β-Fructofuranosidase, Emerging from Nonconventional Codon Usage and Directed Mutation

2010 ◽  
Vol 76 (22) ◽  
pp. 7491-7499 ◽  
Author(s):  
Miguel Álvaro-Benito ◽  
Miguel de Abreu ◽  
Francisco Portillo ◽  
Julia Sanz-Aparicio ◽  
María Fernández-Lobato
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Transferase Activity ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Schwanniomyces Occidentalis ◽  
Directed Mutation

ABSTRACT Schwanniomyces occidentalis β-fructofuranosidase (Ffase) releases β-fructose from the nonreducing ends of β-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (k cat/Km ) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymes' specificity for 6-kestose synthesis. The influence of 6 Ffase residues on enzyme activity was analyzed on both the Leu196/Ser196 backgrounds (Trp47, Asn49, Asn52, Ser111, Lys181, and Pro232). Only N52S and P232V mutations improved the transferase activity of the wild-type enzyme (about 1.6-fold). Modeling the transfructosylation products into the active site, in combination with an analysis of the kinetics and transfructosylation reactions, defined a new region responsible for the transferase specificity of the enzyme.

scholarly journals Crystal structure of human anterior gradient protein 3

2018 ◽  
Vol 74 (7) ◽  
pp. 425-430 ◽  
Author(s):  
Van Dat Nguyen ◽  
Ekaterina Biterova ◽  
Mikko Salin ◽  
Rik K. Wierenga ◽  
Lloyd W. Ruddock
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Recombinant Expression ◽  
Sequence Similarity ◽  
Family Members ◽  
Oxidative Protein Folding ◽  
Active Site Motif ◽  
Share Sequence Similarity ◽  
Protein Disulfide ◽  
Human Family

Oxidative protein folding in the endoplasmic reticulum is catalyzed by the protein disulfide isomerase family of proteins. Of the 20 recognized human family members, the structures of eight have been deposited in the PDB along with domains from six more. Three members of this family, ERp18, anterior gradient protein 2 (AGR2) and anterior gradient protein 3 (AGR3), are single-domain proteins which share sequence similarity. While ERp18 has a canonical active-site motif and is involved in native disulfide-bond formation, AGR2 and AGR3 lack elements of the active-site motif found in other family members and may both interact with mucins. In order to better define its function, the structure of AGR3 is required. Here, the recombinant expression, purification, crystallization and crystal structure of human AGR3 are described.

scholarly journals Molecular Dynamics Simulations Study of the Interactions between Human Dipeptidyl-Peptidase III and Two Substrates

Molecules ◽  
2021 ◽  
Vol 26 (21) ◽  
pp. 6492
Author(s):  
Shitao Zhang ◽  
Shuai Lv ◽  
Xueqi Fu ◽  
Lu Han ◽  
Weiwei Han ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Conformational Changes ◽  
Biological Activities ◽  
Catalytic Efficiency ◽  
Dipeptidyl Peptidase ◽  
Pain Modulation ◽  
Ang Ii ◽  
Α Helix ◽  
Dynamics Simulations

Human dipeptidyl-peptidase III (hDPP III) is capable of specifically cleaving dipeptides from the N-terminal of small peptides with biological activity such as angiotensin II (Ang II, DRVYIHPF), and participates in blood pressure regulation, pain modulation, and the development of cancers in human biological activities. In this study, 500 ns molecular dynamics simulations were performed on free-hDPP III (PDB code: 5E33), hDPP III–Ang II (PDB code: 5E2Q), and hDPP III–IVYPW (PDB code: 5E3C) to explore how these two peptides affect the catalytic efficiency of enzymes in terms of the binding mode and the conformational changes. Our results indicate that in the case of the hDPP III–Ang II complex, subsite S1 became small and hydrophobic, which might be propitious for the nucleophile to attack the substrate. The structures of the most stable conformations of the three systems revealed that Arg421-Lys423 could form an α-helix with the presence of Ang II, but only part of the α-helix was produced in hDPP III-IVYPW. As the hinge structure in hDPP III, the conformational changes that took place in the Arg421-Lys423 residue could lead to the changes in the shape and space of the catalytic subsites, which might allow water to function as a nucleophile to attack the substrate. Our results may provide new clues to enable the design of new inhibitors for hDPP III in the future.

scholarly journals Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nur Suhanawati Ashaari ◽  
Mohd Hairul Ab. Rahim ◽  
Suriana Sabri ◽  
Kok Song Lai ◽  
Adelene Ai-Lian Song ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Homology Model ◽  
Biochemical Properties ◽  
Terpene Synthase ◽  
Monoterpene Synthase ◽  
Terminal Domain ◽  
Catalytic Active Site ◽  
Plectranthus Amboinicus

AbstractLinalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

scholarly journals Correction: Dynamic properties of dipeptidyl peptidase III from Bacteroides thetaiotaomicron and the structural basis for its substrate specificity – a computational study

2017 ◽  
Vol 13 (12) ◽  
pp. 2729-2730
Author(s):  
M. Tomin ◽  
S. Tomić
Keyword(s):  
Substrate Specificity ◽  
Computational Study ◽  
Dynamic Properties ◽  
Dipeptidyl Peptidase ◽  
Structural Basis ◽  
Bacteroides Thetaiotaomicron

Correction for ‘Dynamic properties of dipeptidyl peptidase III from Bacteroides thetaiotaomicron and the structural basis for its substrate specificity – a computational study’ by M. Tomin et al., Mol. BioSyst., 2017, 13, 2407–2417.

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