scholarly journals Sequence of human carboxypeptidase D reveals it to be a member of the regulatory carboxypeptidase family with three tandem active site domains

1997 ◽  
Vol 327 (1) ◽  
pp. 81-87 ◽  
Author(s):  
Fulong TAN ◽  
Michael REHLI ◽  
Stefan W. KRAUSE ◽  
Randal A. SKIDGEL
Keyword(s):  
Active Site ◽  
Sequence Similarity ◽  
Northern Blotting ◽  
Active Site Residues ◽  
Virus Binding ◽  
Ph Optimum ◽  
Hydrophobic Region ◽  
Reading Frame ◽  
Carboxypeptidase D ◽  
Domain 3

We have cloned the cDNA for human carboxypeptidase D (CPD), a new B-type metallocarboxypeptidase that is membrane bound and has an acidic pH optimum. The 5.8 kb of cDNA sequenced contains an open reading frame of 4131 bp encoding 1377 amino acid residues. The sequence is similar (75% identity) to duck gp180, a protein that was isolated, cloned and sequenced as a hepatitis B virus-binding protein but not characterized as a carboxypeptidase. Hydropathic analysis revealed a hydrophobic region at the N-terminus, representing the signal peptide, and one near the C-terminus that probably represents the transmembrane anchor. The most striking feature is the presence of three tandem carboxypeptidase homology domains that have sequence similarity to the regulatory B-type carboxypeptidase family, typified by carboxypeptidases M, E and N. Because of the three repeats, CPD is about three times larger (175–180 kDa) than other members of this family (approx. 50–62 kDa). Domain 2 is most closely related to carboxypeptidases M, E and N (45–48% identity), followed by domain 1 (37–38%) and domain 3 (20–27%). There is much higher sequence identity in regions containing putative active site residues, and all catalytically important residues are strictly conserved in domains 1 and 2. In domain 3, however, only 1 of 8 active site residues is conserved, indicating that this portion might not be catalytically active. Northern blotting of mRNA from human tissues and cells showed high levels of CPD mRNA in placenta, pancreas and Hep G2 hepatoma cells, and smaller amounts in skeletal muscle, heart and HT-29 colon carcinoma and melanoma cell lines.

Structural analysis of mycobacterial branched-chain aminotransferase: implications for inhibitor design

2010 ◽  
Vol 66 (5) ◽  
pp. 549-557 ◽  
Author(s):  
Alina Castell ◽  
Christian Mille ◽  
Torsten Unge
Keyword(s):  
Active Site ◽  
Sequence Similarity ◽  
Specific Inhibitor ◽  
Inhibition Assay ◽  
Active Site Residues ◽  
X Ray ◽  
Sequence Identity ◽  
Branched Chain ◽  
Functional Analyses ◽  
Branched Chain Aminotransferase

The branched-chain aminotransferase (BCAT) ofMycobacterium tuberculosishas been characterized as being essential to the survival of the bacterium. The enzyme is pyridoxal 5′-phosphate-dependent and belongs to the aminotransferase IIIa subfamily, to which the human BCATs also belong. The overall sequence similarity is high within the subfamily and the sequence identity among the active-site residues is high. In order to identify structurally unique features ofM. tuberculosisBCAT, X-ray structural and functional analyses of the closely related BCAT fromM. smegmatiswere carried out. The crystal structures include the apo form at 2.2 Å resolution and a 1.9 Å structure of the holo form cocrystallized with the inhibitorO-benzylhydroxylamine (Obe). The analyses highlighted the active-site residues Tyr209 and Gly243 as being structurally unique characteristics of the mycobacterial BCATs relative to the human BCATs. The inhibitory activities of Obe and ammonium sulfate were verified in an inhibition assay. Modelling of the inhibitor Obe in the substrate pocket indicated potential for the design of a mycobacterial-specific inhibitor.

scholarly journals A soluble ACE2 microbody protein fused to a single immunoglobulin Fc domain is a potent inhibitor of SARS-CoV-2 infection in cell culture

2020 ◽  
Author(s):  
Takuya Tada ◽  
Chen Fan ◽  
Ramanjit Kaur ◽  
Kenneth A. Stapleford ◽  
Harry Gristick ◽  
...  
Keyword(s):  
Cell Culture ◽  
Active Site ◽  
Fusion Proteins ◽  
Potent Inhibitor ◽  
Spike Protein ◽  
Pseudotyped Virus ◽  
Virus Binding ◽  
Catalytic Active Site ◽  
Domain 3 ◽  
High Infectivity

SummarySoluble forms of ACE2 have recently been shown to inhibit SARS-CoV-2 infection. We report on an improved soluble ACE2, termed a “microbody” in which the ACE2 ectodomain is fused to Fc domain 3 of the immunoglobulin heavy chain. The protein is smaller than previously described ACE2-Ig Fc fusion proteins and contains an H345A mutation in the ACE2 catalytic active site that inactivates the enzyme without reducing its affinity for the SARS-CoV-2 spike. The disulfide-bonded ACE2 microbody protein inhibited entry of lentiviral SARS-CoV-2 spike protein pseudotyped virus and live SARS-CoV-2 with a potency 10-fold higher than unmodified soluble ACE2 and was active after initial virus binding to the cell. The ACE2 microbody inhibited the entry of ACE2-specific β coronaviruses and viruses with the high infectivity variant D614G spike. The ACE2 microbody may be a valuable therapeutic for COVID-19 that is active against SARS-CoV-2 variants and future coronaviruses that may arise.

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 Structural and Functional Analysis of Rv3214 from Mycobacterium tuberculosis, a Protein with Conflicting Functional Annotations, Leads to Its Characterization as a Phosphatase

2006 ◽  
Vol 188 (10) ◽  
pp. 3589-3599 ◽  
Author(s):  
Harriet A. Watkins ◽  
Edward N. Baker
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Functional Annotation ◽  
Sequence Similarity ◽  
Phosphoglycerate Mutase ◽  
Phosphopantetheinyl Transferase ◽  
Ph Optimum ◽  
Reading Frame ◽  
Functional Annotations ◽  
The Many

ABSTRACT The availability of complete genome sequences has highlighted the problems of functional annotation of the many gene products that have only limited sequence similarity with proteins of known function. The predicted protein encoded by open reading frame Rv3214 from the Mycobacterium tuberculosis H37Rv genome was originally annotated as EntD through sequence similarity with the Escherichia coli EntD, a 4′-phosphopantetheinyl transferase implicated in siderophore biosynthesis. An alternative annotation, based on slightly higher sequence identity, grouped Rv3214 with proteins of the cofactor-dependent phosphoglycerate mutase (dPGM) family. The crystal structure of this protein has been solved by single-wavelength anomalous dispersion methods and refined at 2.07-Å resolution (R = 0.229; Rfree = 0.245). The protein is dimeric, with a monomer fold corresponding to the classical dPGM α/β structure, albeit with some variations. Closer comparisons of structure and sequence indicate that it most closely corresponds with a broad-spectrum phosphatase subfamily within the dPGM superfamily. This functional annotation has been confirmed by biochemical assays which show negligible mutase activity but acid phosphatase activity with a pH optimum of 5.4 and suggests that Rv3214 may be important for mycobacterial phosphate metabolism in vivo. Despite its weak sequence similarity with the 4′-phosphopantetheinyl transferases (EntD homologues), there is little evidence to support this function.

scholarly journals Cloning and characterization of a novel nuclease from shrimp hepatopancreas, and prediction of its active site

2000 ◽  
Vol 346 (3) ◽  
pp. 799-804 ◽  
Author(s):  
Wen-Yi WANG ◽  
Shwu-Huey LIAW ◽  
Ta-Hsiu LIAO
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Sequence Similarity ◽  
Distinct Group ◽  
Structural Data ◽  
Hydrolytic Activity ◽  
Degenerate Primers ◽  
Homologous Proteins ◽  
Reading Frame

Approximately 95% of the amino acid sequence of a shrimp (Penaeus japonicus) nuclease was derived from protease-digested peptides. A 1461-base cDNA for the nuclease was amplified and sequenced with degenerate primers based on the amino acid sequence and then specific primers by 3ʹ and 5ʹ RACE (rapid amplification of cDNA ends). It contains an open reading frame encoding a putative 21-residue signal peptide and a 381-residue mature protein. The N-terminus of the enzyme is pyroglutamate, deduced from composition and matrix-assisted laser desorption ionization-time-of-flight MS analyses, and confirmed by a glutamine residue in the cDNA sequence. The enzyme has 11 Cys residues, forming five intramolecular disulphides. The eleventh Cys residue was linked to a thiol compound with an estimated molecular mass of between 500 and 700 Da. A sequence similarity search revealed no homologous proteins but residues 205-255 shared a conserved active-site motif within a distinct group of nucleases. His211 in this conserved motif was shown to be very important in catalysis by site-specific modification with 14C-labelled iodoacetate. The shrimp nuclease, previously designated DNase I, does indeed possess a low level of hydrolytic activity towards RNA in the presence of Mg2+ and Ca2+. The conservation of functionally important residues during distant evolution might imply that the catalytic mechanisms are similar in these nucleases, which should be classified in one subfamily. Finally, an active-site structure for shrimp nuclease was proposed on the basis of published structural data and the results of mutational and biochemical analyses of Serratia nuclease.

scholarly journals Regulation of carboxypeptidase E. Effect of pH, temperature and Co2+ on kinetic parameters of substrate hydrolysis

1992 ◽  
Vol 285 (2) ◽  
pp. 613-618 ◽  
Author(s):  
D Greene ◽  
B Das ◽  
L D Fricker
Keyword(s):  
Active Site ◽  
Bound Water ◽  
Active Site Residues ◽  
Carboxypeptidase A ◽  
Ph Optimum ◽  
Ph Profile ◽  
Site Model ◽  
Carboxypeptidase E ◽  
The Difference ◽  
Substrate Hydrolysis

Carboxypeptidase E is a member of the carboxypeptidase A and B gene family, with many of the putative active-site and substrate-binding residues conserved between these enzymes. However, the pH optimum of carboxypeptidase E is substantially lower than that of carboxypeptidases A and B. To evaluate whether the difference in the pH optima of these carboxypeptidases reflects fundamental differences in the ionization behaviour of active-site residues, the influence of pH on carboxypeptidase E activity was examined. The V(max) for hydrolysis of dansyl-Phe-Ala-Arg is pH-independent between 5 and 7, but decreases at pH values below 5. The pKa for the group the protonation of which leads to the loss of activity is approximately 4.8, and the slope of the V(max.)/pH profile suggests that only a single ionizable group is involved. In contrast, Km and V(max.)/Km are dramatically influenced by pH over the range 5-7, with multiple ionizable groups detected in this pH range. The pKa of the group the protonation of which decreases the V(max.) of substrate hydrolysis is lower (4.5) for carboxypeptidase E which had been reconstituted with Co2+. The enthalpy of ionization of the group observed in the V(max.) profile for carboxypeptidase E is approx. 28.9 kJ/mol. These results are compatible with the active-site model of the homologous carboxypeptidase A: in this model the ionization of a metal-bound water molecule is responsible for the observed decrease in V(max.).

scholarly journals Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

2001 ◽  
Vol 67 (2) ◽  
pp. 673-679 ◽  
Author(s):  
Kazuhiko Ishikawa ◽  
Hiroyasu Ishida ◽  
Ikuo Matsui ◽  
Yutaka Kawarabayasi ◽  
Hisasi Kikuchi
Keyword(s):  
Active Site ◽  
Sequence Similarity ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Vector System ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
High Sequence Similarity ◽  
Pyrococcus Horikoshii ◽  
Thermophilic Archaeon

ABSTRACT Genome sequencing of the thermophilic archaeon Pyrococcus horikoshii OT3 revealed a gene which had high sequence similarity to the gene encoding the carboxypeptidase ofSulfolobus solfataricus and also to that encoding the aminoacylase from Bacillus stearothermophilus. The gene from P. horikoshii comprises an open reading frame of 1,164 bp with an ATG initiation codon and a TGA termination codon, encoding a 43,058-Da protein of 387 amino acid residues. However, some of the proposed active-site residues for carboxypeptidase were not found in this gene. The gene was overexpressed in Escherichia coli with the pET vector system, and the expressed enzyme had high hydrolytic activity for both carboxypeptidase and aminoacylase at high temperatures. The enzyme was stable at 90°C, with the highest activity above 95°C. The enzyme contained one bound zinc ion per one molecule that was essential for the activity. The results of site-directed mutagenesis of Glu367, which corresponds to the essential Glu270 in bovine carboxypeptidase A and the essential Glu in other known carboxypeptidases, revealed that Glu367 was not essential for this enzyme. The results of chemical modification of the SH group and site-directed mutagenesis of Cys102 indicated that Cys102 was located at the active site and was related to the activity. From these findings, it was proven that this enzyme is a hyperthermostable, bifunctional, new zinc-dependent metalloenzyme which is structurally similar to carboxypeptidase but whose hydrolytic mechanism is similar to that of aminoacylase. Some characteristics of this enzyme suggested that carboxypeptidase and aminoacylase might have evolved from a common origin.

Development of Xanthine Based Inhibitors Targeting Phosphodiesterase 9A

2017 ◽  
Vol 14 (10) ◽  
pp. 1122-1137 ◽  
Author(s):  
Nivedita Singh ◽  
Parameswaran Saravanan ◽  
M.S. Thakur ◽  
Sanjukta Patra
Keyword(s):  
Free Energy ◽  
Active Site ◽  
Signal Transduction Pathway ◽  
Docking Study ◽  
Primary Objective ◽  
Cgmp Level ◽  
Active Site Residues ◽  
Aromatic Fragment ◽  
Docking Approach ◽  
Free Energy Of Binding

Background: Phosphodiesterases 9A (PDE9A) is one of the prominent regulating enzymes of the signal transduction pathway having highest catalytic affinity for second messenger, cGMP. When the cGMP level is lowered, an uncontrolled expression of PDE9A may lead to various neurodegenerative diseases. To regulate the catalytic activity of PDE9A, potent inhibitors are needed. Objective: The primary objective of the present study was to develop new xanthine based inhibitors targeting PDE9A. This study was an attempt to bring structural diversification in PDE9A inhibitor development because most of the existing inhibitors are constructed over pyrazolopyrimidinone scaffold. Methods: Manual designing and parallel molecular docking approach were used for the development of xanthine derivatives. In this study, N1, N3, N9 and C8 positions of xanthine scaffold were selected as substitution sites to design 200 new compounds. Reverse docking and pharmaceutical analyses were used for final validation of most promising compounds. Results: By keeping free energy of binding cut-off of -6.0 kcal/mol, 52 compounds were screened. The compounds with substitution at N1, N3 and C8 positions of xanthine showed good occupancy in PDE9A active site pocket with a significant interaction pattern. This was further validated by screening different factors such as free energy of binding, inhibition constant and interacting active site residues in the 5Å region. Substitution at C8 position with phenyl substituent determined the inhibition affinity of compounds towards PDE9A by establishing a strong hydrophobic - hydrophobic interaction. The alkyl chain at N1 position generated selectivity of compounds towards PDE9A. The aromatic fragment at N3 position increased the binding affinity of compounds. Thus, by comparative docking study, it was found that compound 39-42 formed selective interaction towards PDE9A over other members of the PDE superfamily. Conclusion: From the present study, N1, N3 and C8 positions of xanthine were concluded as the best sites for substitution for the generation of potent PDE9A inhibitors.

scholarly journals An enzymatic activation of formaldehyde for nucleotide methylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Charles Bou-Nader ◽  
Frederick W. Stull ◽  
Ludovic Pecqueur ◽  
Philippe Simon ◽  
Vincent Guérineau ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thymidylate Synthase ◽  
Active Site ◽  
De Novo ◽  
Crystallographic Structure ◽  
De Novo Synthesis ◽  
Active Site Residues ◽  
Enzymatic Activation ◽  
Enzyme Cofactors ◽  
Unique Ability

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

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