scholarly journals Dipeptidyl Aminopeptidase IV from Stenotrophomonas maltophilia Exhibits Activity against a Substrate Containing a 4-Hydroxyproline Residue

2008 ◽  
Vol 190 (23) ◽  
pp. 7819-7829 ◽  
Author(s):  
Yoshitaka Nakajima ◽  
Kiyoshi Ito ◽  
Tsubasa Toshima ◽  
Takashi Egawa ◽  
Heng Zheng ◽  
...  
Keyword(s):  
Active Site ◽  
Stenotrophomonas Maltophilia ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Subunit Structure ◽  
Hydrophobic Pocket ◽  
Cell Parameters ◽  
Replacement Method ◽  
Major Factors ◽  
Dipeptidyl Aminopeptidase

ABSTRACT The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-Å resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P43212, with unit cell parameters a = b = 105.9 Å and c = 161.9 Å. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal β-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.

scholarly journals Disruption of the crossover helix impairs dihydrofolate reductase activity in the bifunctional enzyme TS–DHFR from Cryptosporidium hominis

2009 ◽  
Vol 417 (3) ◽  
pp. 757-764 ◽  
Author(s):  
Melissa A. Vargo ◽  
W. Edward Martucci ◽  
Karen S. Anderson
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Reductase Activity ◽  
The Other ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Bifunctional Enzyme ◽  
Cryptosporidium Hominis ◽  
Single Polypeptide Chain ◽  
Species Specific

In contrast with most species, including humans, which have monofunctional forms of the folate biosynthetic enzymes TS (thymidylate synthase) and DHFR (dihydrofolate reductase), several pathogenic protozoal parasites, including Cryptosporidium hominis, contain a bifunctional form of the enzymes on a single polypeptide chain having both catalytic activities. The crystal structure of the bifunctional enzyme TS–DHFR C. hominis reveals a dimer with a ‘crossover helix’, a swap domain between DHFR domains, unique in that this helical region from one monomer makes extensive contacts with the DHFR active site of the other monomer. In the present study, we used site-directed mutagenesis to probe the role of this crossover helix in DHFR catalysis. Mutations were made to the crossover helix: an ‘alanine-face’ enzyme in which the residues on the face of the helix close to the DHFR active site of the other subunit were mutated to alanine, a ‘glycine-face’ enzyme in which the same residues were mutated to glycine, and an ‘all-alanine’ helix in which all residues of the helix were mutated to alanine. These mutant enzymes were studied using a rapid transient kinetic approach. The mutations caused a dramatic decrease in the DHFR activity. The DHFR catalytic activity of the alanine-face mutant enzyme was 30 s−1, the glycine-face mutant enzyme was 17 s−1, and the all-alanine helix enzyme was 16 s−1, all substantially impaired from the wild-type DHFR activity of 152 s−1. It is clear that loss of helix interactions results in a marked decrease in DHFR activity, supporting a role for this swap domain in DHFR catalysis. The crossover helix provides a unique structural feature of C. hominis bifunctional TS–DHFR that could be exploited as a target for species-specific non-active site inhibitors.

scholarly journals Conversion of citrate synthase into citryl-CoA lyase as a result of mutation of the active-site aspartic acid residue to glutamic acid

1991 ◽  
Vol 280 (2) ◽  
pp. 521-526 ◽  
Author(s):  
W J Man ◽  
Y Li ◽  
C D O'Connor ◽  
D C Wilton
Keyword(s):  
Catalytic Activity ◽  
Aspartic Acid ◽  
Active Site ◽  
Citrate Synthase ◽  
Condensation Reaction ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Reverse Direction ◽  
Acetyl Coa ◽  
Aspartic Acid Residue

The active-site aspartic acid residue, Asp-362, of Escherichia coli citrate synthase was changed by site-directed mutagenesis to Glu-362, Asn-362 or Gly-362. Only very low catalytic activity could be detected with the Asp→Asn and Asp→Gly mutations. The Asp→Glu mutation produced an enzyme that expressed about 0.8% of the overall catalytic rate, and the hydrolysis step in the reaction, monitored as citryl-CoA hydrolysis, was inhibited to a similar extent. However, the condensation reaction, measured in the reverse direction as citryl-CoA cleavage to oxaloacetate and acetyl-CoA, was not affected by the mutation, and this citryl-CoA lyase activity was the major catalytic activity of the mutant enzyme. This high condensation activity in an enzyme in which the subsequent hydrolysis step was about 98% inhibited permitted considerable exchange of the methyl protons of acetyl-CoA during catalysis by the mutant enzyme. The Km for oxaloacetate was not significantly altered in the D362E mutant enzyme, whereas the Km for acetyl-CoA was about 5 times lower. A mechanism is proposed in which Asp-362 is involved in the hydrolysis reaction of this enzyme, and not as a base in the deprotonation of acetyl-CoA as recently suggested by others. [Karpusas, Branchaud & Remington (1990) Biochemistry 29, 2213-2219; Alter, Casazza, Zhi, Nemeth, Srere & Evans, (1990) Biochemistry 29, 7557-7563].

scholarly journals Periplasmic form of dipeptidyl aminopeptidase IV fromPseudoxanthomonas mexicanaWO24: purification, kinetic characterization, crystallization and X-ray crystallographic analysis

2017 ◽  
Vol 73 (11) ◽  
pp. 601-606 ◽  
Author(s):  
Saori Roppongi ◽  
Chika Tateoka ◽  
Mayu Fujimoto ◽  
Ippei Iizuka ◽  
Saori Morisawa ◽  
...  
Keyword(s):  
Crystal Form ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Method ◽  
Dpp Iv ◽  
Molecular Replacement Method ◽  
Dipeptidyl Aminopeptidase

Dipeptidyl aminopeptidase IV (DAP IV or DPP IV) fromPseudoxanthomonas mexicanaWO24 (PmDAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position [NH2-P2-P1(Pro/Ala)-P1′-P2′…]. For crystallographic studies, the periplasmic form of PmDAP IV was overproduced inEscherichia coli, purified and crystallized in complex with the tripeptide Lys-Pro-Tyr using the hanging-drop vapour-diffusion method. Kinetic parameters of the purified enzyme against a synthetic substrate were also determined. X-ray diffraction data to 1.90 Å resolution were collected from a triclinic crystal form belonging to space groupP1, with unit-cell parametersa= 88.66,b= 104.49,c = 112.84 Å, α = 67.42, β = 68.83, γ = 65.46°. Initial phases were determined by the molecular-replacement method usingStenotrophomonas maltophiliaDPP IV (PDB entry 2ecf) as a template and refinement of the structure is in progress.

scholarly journals Purification and Biochemical Characterization of Taxadiene Synthase from Bacillus koreensis and Stenotrophomonas maltophilia

2021 ◽  
Vol 89 (4) ◽  
pp. 48
Author(s):  
Ashraf S. A. El-Sayed ◽  
Maher Fathalla ◽  
Ahmed A. Shindia ◽  
Amgad M. Rady ◽  
Ashraf F. El-Baz ◽  
...  
Keyword(s):  
Active Site ◽  
Stenotrophomonas Maltophilia ◽  
Genomic Feature ◽  
Subunit Structure ◽  
Fragmentation Pattern ◽  
Intermediary Metabolites ◽  
Taxadiene Synthase ◽  
Taxol Biosynthesis ◽  
Main Challenge

Taxadiene synthase (TDS) is the rate-limiting enzyme of Taxol biosynthesis that cyclizes the geranylgeranyl pyrophosphate into taxadiene. Attenuating Taxol productivity by fungi is the main challenge impeding its industrial application; it is possible that silencing the expression of TDS is the most noticeable genomic feature associated with Taxol-biosynthetic abolishing in fungi. As such, the characterization of TDS with unique biochemical properties and autonomous expression that is independent of transcriptional factors from the host is the main challenge. Thus, the objective of this study was to kinetically characterize TDS from endophytic bacteria isolated from different plants harboring Taxol-producing endophytic fungi. Among the recovered 23 isolates, Bacillus koreensis and Stenotrophomonas maltophilia achieved the highest TDS activity. Upon using the Plackett–Burman design, the TDS productivity achieved by B. koreensis (18.1 µmol/mg/min) and S. maltophilia (14.6 µmol/mg/min) increased by ~2.2-fold over the control. The enzyme was purified by gel-filtration and ion-exchange chromatography with ~15 overall folds and with molecular subunit structure 65 and 80 kDa from B. koreensis and S. maltophilia, respectively. The chemical identity of taxadiene was authenticated from the GC-MS analyses, which provided the same mass fragmentation pattern of authentic taxadiene. The tds gene was screened by PCR with nested primers of the conservative active site domains, and the amplicons were sequenced, displaying a higher similarity with tds from T. baccata and T. brevifolia. The highest TDS activity by both bacterial isolates was recorded at 37–40 °C. The Apo-TDSs retained ~50% of its initial holoenzyme activities, ensuring their metalloproteinic identity. The activity of purified TDS was completely restored upon the addition of Mg2+, confirming the identity of Mg2+ as a cofactor. The TDS activity was dramatically reduced upon the addition of DTNB and MBTH, ensuring the implementation of cysteine-reactive thiols and ammonia groups on their active site domains. This is the first report exploring the autonomous robust expression TDS from B. koreensis and S. maltophilia with a higher affinity to cyclize GGPP into taxadiene, which could be a novel platform for taxadiene production as intermediary metabolites of Taxol biosynthesis.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

Active amino acids of the Kell blood group protein and model of the ectodomain based on the structure of neutral endopeptidase 24.11

Blood ◽  
2003 ◽  
Vol 102 (8) ◽  
pp. 3028-3034 ◽  
Author(s):  
Soohee Lee ◽  
Asim K. Debnath ◽  
Colvin M. Redman
Keyword(s):  
Amino Acids ◽  
Blood Group ◽  
Active Site ◽  
Neutral Endopeptidase ◽  
Site Directed Mutagenesis ◽  
Peptide Hydrolysis ◽  
Enzyme Active Site ◽  
Endopeptidase 24.11 ◽  
Outer Domain ◽  
Group Protein

Abstract In addition to its importance in transfusion, Kell protein is a member of the M13 family of zinc endopeptidases and functions as an endothelin-3–converting enzyme. To obtain information on the structure of Kell protein we built a model based on the crystal structure of the ectodomain of neutral endopeptidase 24.11 (NEP). Similar to NEP, the Kell protein has 2 globular domains consisting mostly of α-helical segments. The domain situated closest to the membrane contains both the N- and C-terminal sequences and the enzyme-active site. The outer domain contains all of the amino acids whose substitutions lead to different Kell blood group phenotypes. In the model, the zinc peptidase inhibitor, phosphoramidon, was docked in the active site. Site-directed mutagenesis of amino acids in the active site was performed and the enzymatic activities of expressed mutant Kell proteins analyzed and compared with NEP. Our studies indicate that Kell and NEP use the same homologous amino acids in the coordination of zinc and in peptide hydrolysis. However, Kell uses different amino acids than NEP in substrate binding and appears to have more flexibility in the composition of amino acids allowed in the active site.

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