scholarly journals Structural Insight into a Yeast Maltase—The BaAG2 from Blastobotrys adeninivorans with Transglycosylating Activity

2021 ◽  
Vol 7 (10) ◽  
pp. 816
Author(s):  
Karin Ernits ◽  
Christian Kjeldsen ◽  
Karina Persson ◽  
Eliis Grigor ◽  
Tiina Alamäe ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Temperature Tolerance ◽  
Catalytic Triad ◽  
Arxula Adeninivorans ◽  
Transglycosylation Activity ◽  
Potential Binding ◽  
Active Site Cleft ◽  
Prebiotic Oligosaccharides

An early-diverged yeast, Blastobotrys (Arxula) adeninivorans (Ba), has biotechnological potential due to nutritional versatility, temperature tolerance, and production of technologically applicable enzymes. We have biochemically characterized from the Ba type strain (CBS 8244) the GH13-family maltase BaAG2 with efficient transglycosylation activity on maltose. In the current study, transglycosylation of sucrose was studied in detail. The chemical entities of sucrose-derived oligosaccharides were determined using nuclear magnetic resonance. Several potentially prebiotic oligosaccharides with α-1,1, α-1,3, α-1,4, and α-1,6 linkages were disclosed among the products. Trisaccharides isomelezitose, erlose, and theanderose, and disaccharides maltulose and trehalulose were dominant transglycosylation products. To date no structure for yeast maltase has been determined. Structures of the BaAG2 with acarbose and glucose in the active center were solved at 2.12 and 2.13 Å resolution, respectively. BaAG2 exhibited a catalytic domain with a (β/α)8-barrel fold and Asp216, Glu274, and Asp348 as the catalytic triad. The fairly wide active site cleft contained water channels mediating substrate hydrolysis. Next to the substrate-binding pocket an enlarged space for potential binding of transglycosylation acceptors was identified. The involvement of a Glu (Glu309) at subsite +2 and an Arg (Arg233) at subsite +3 in substrate binding was shown for the first time for α-glucosidases.

THE CATALYTIC TRIAD RESIDUES (HIS221, ASP269, SER365) AND THE BINDING POCKET RESIDUE (ASP359) IN FACTOR IXBm ELSINORE (IXBmLE) ARE NOT ALTERED

1987 ◽  
Author(s):  
S G Spitzer ◽  
P Usharani ◽  
A D Roser ◽  
C K Kasper ◽  
S G Bajaj
Keyword(s):  
Catalytic Domain ◽  
Antithrombin Iii ◽  
Genomic Library ◽  
Indirect Evidence ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
Factor Vii ◽  
Factor X ◽  
Liver Cdna Library ◽  
Synthetic Oligonucleotides

Previous studies suggested that the defect in IXBmLE (a nonfunctional variant of human IX) is either in the catalytic triad or at the site(s) of interaction with the macromolecular substrates (antithrombin III, factor VII or factor X). To distinguish between these possibilities, we isolated a complete IX cDNA clone from a human liver cDNA library. We also constructed a genomic library (in phage EMBL3) using DNA of the BmLE patient. The library was screened with normal IX cDNA and with synthetic oligonucleotides. The positive clones containing the exons coding for IX were plaque purified. Two clones which contained the coding sequence of the catalytic domain, i.e., His221 (exon VII), and Asp269, Asp359, and Ser365 (exon VIII) were selected for further studies. The phage containing exon VIII was first digested with Sail and EcoRI and a 2-Kb fragment, which hybridized with the segment of cDNA containing exon VIII, was gel purified. The 2-Kb fragment was further digested and the subfragments were cloned into M13; the length and direction of the fragments used in sequencing are shown below:The phage containing exon VII was digested with PstI and SalI, and a 1-Kb fragment that hybridized with the 19-mer His221 probe was subcloned into M13 phage for sequencing. The sequence starting with residue Vall96 through residue Arg403 was found to be normal. Thus, none of the residues in the catalytic domain of IXBmLE are different from that of normal IX. These data provide strong indirect evidence that the noncatalytic aminoterminal portion of IX plays a significant role in the structural recognition of the macromolecular substrates. The sequence of this region of IXBmLE should provide information about the putative residue(s) essential for this recognition.

Structural and Functional Basis of Plasminogen Activation by Staphylokinase

1999 ◽  
Vol 81 (04) ◽  
pp. 479-485 ◽  
Author(s):  
L. Jespers ◽  
S. Vanwetswinkel ◽  
H. R. Lijnen ◽  
N. Van Herzeele ◽  
B. Van Hoef ◽  
...  
Keyword(s):  
Active Site ◽  
Recombinant Dna ◽  
Catalytic Domain ◽  
Functional Model ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
X Ray Crystallography ◽  
Serine Protease Domain ◽  
Docking Model ◽  
Binding Epitope

SummaryStaphylokinase (Sak), a 15.5-kDa bacterial protein, forms a complex with human plasmin, which in turn activates other plasminogen molecules to plasmin. Three recombinant DNA-based approaches, (i) site directed substitution with alanine, (ii) search for proximity relationships at the complex interface, and (iii) active-site accessibility to protease inhibitors have been used to deduce a coherent docking model of the crystal structure of Sak on the homology-based model of micro-plasmin (μPli), the serine protease domain of plasmin. Sak binding on μPli is primarily mediated by two surface-exposed loops, loops 174 and 215, at the rim of the active-site cleft, while the binding epitope of Sak on μPli involves several residues located in the flexible NH2-terminal arm and in the five-stranded mixed β-sheet. Several Sak residues located within the unique μ-helix and the β2 strand do not contribute to the binding epitope but are essential to induce plasminogen activating potential in the Sak:μPli complex. These residues form a topologically distinct activation epitope, which, upon binding of Sak to the catalytic domain of μPli, protrudes into a broad groove near the catalytic triad of μPli, thereby generating a competent binding pocket for micro-plasminogen (μPlg), which buries approximately 2500 Å of the Sak:μPli complex upon binding. This structural and functional model may serve as a template for the design of improved Sak-derived thrombolytic agents. Following the completion and presentation of the present study, the deduced Sak:μPli:μPlg complex was fully confirmed by X-ray crystallography, which further illustrates the power and potential of the present approach.

scholarly journals Structural analysis of the catalytic domain of Artemis endonuclease/SNM1C reveals distinct structural features

2020 ◽  
Vol 295 (35) ◽  
pp. 12368-12377 ◽  
Author(s):  
Md Fazlul Karim ◽  
Shanshan Liu ◽  
Adrian R. Laciak ◽  
Leah Volk ◽  
Mary Koszelak-Rosenblum ◽  
...  
Keyword(s):  
B Cell ◽  
Catalytic Domain ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Structural Features ◽  
Zinc Ion ◽  
Dna Double Strand Breaks ◽  
Repair Gene ◽  
Dna Hairpins ◽  
Binding Surface

The endonuclease Artemis is responsible for opening DNA hairpins during V(D)J recombination and for processing a subset of pathological DNA double-strand breaks. Artemis is an attractive target for the development of therapeutics to manage various B cell and T cell tumors, because failure to open DNA hairpins and accumulation of chromosomal breaks may reduce the proliferation and viability of pre-T and pre-B cell derivatives. However, structure-based drug discovery of specific Artemis inhibitors has been hampered by a lack of crystal structures. Here, we report the structure of the catalytic domain of recombinant human Artemis. The catalytic domain displayed a polypeptide fold similar overall to those of other members in the DNA cross-link repair gene SNM1 family and in mRNA 3′-end-processing endonuclease CPSF-73, containing metallo-β-lactamase and β-CASP domains and a cluster of conserved histidine and aspartate residues capable of binding two metal atoms in the catalytic site. As in SNM1A, only one zinc ion was located in the Artemis active site. However, Artemis displayed several unique features. Unlike in other members of this enzyme class, a second zinc ion was present in the β-CASP domain that leads to structural reorientation of the putative DNA-binding surface and extends the substrate-binding pocket to a new pocket, pocket III. Moreover, the substrate-binding surface exhibited a dominant and extensive positive charge distribution compared with that in the structures of SNM1A and SNM1B, presumably because of the structurally distinct DNA substrate of Artemis. The structural features identified here may provide opportunities for designing selective Artemis inhibitors.

Characterization of the extracellular poly(3-hydroxybutyrate) depolymerase ofComamonassp. and of its structural gene

1995 ◽  
Vol 41 (13) ◽  
pp. 160-169 ◽  
Author(s):  
Dieter Jendrossek ◽  
Martina Backhaus ◽  
Meike Andermann
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Binding Site ◽  
Catalytic Domain ◽  
Substrate Binding ◽  
Catalytic Triad ◽  
Reading Frame ◽  
Phb Depolymerase ◽  
Substrate Binding Site

The poly(3-hydroxybutyrate) (PHB) depolymerase structural gene of Comamonas sp. (phaZCsp) was cloned in Escherichia coli and identified by halo formation on PHB-containing solid medium. The nucleotide sequence of a 1719 base pair MboI fragment was determined and contained one large open reading frame (ORF1, 1542 base pairs). This open reading frame encoded the precursor of the PHB depolymerase (514 amino acids; Mr, 53 095), and the deduced amino acid sequence was in agreement with the N-terminal amino acid sequence of the purified PHB depolymerase from amino acid 26 onwards. Analysis of the deduced amino acid sequence revealed a domain structure of the protein: a signal peptide that was 25 amino acids long was followed by a catalytic domain of about 300 amino acids, a fibronectin type III (Fn3) modul sequence, and a putative PHB-specific substrate-binding site. By comparison of the primary structure with that of other polyhydroxyalkanoate (PHA) depolymerases, the catalytic domain apparently contained a catalytic triad of serine, histidine, and aspartate. In addition, a conserved region resembling the oxyanion hole of lipases was present. The catalytic domain was linked to a C-terminal putative substrate-binding site by a sequence about 90 amino acids long resembling the Fn3 modul of fibronectin and other eukaryotic extracellular matrix proteins. A threonine-rich region, which was found in four of five PHA depolymerases of Pseudomonas lemoignei, was not present in the Comamonas sp. depolymerase. The similarities with and differences from other PHA depolymerases are discussed.Key words: biodegradable polymer, poly(3-hydroxybutyrate) depolymerase, serine hydrolase, catalytic triad, Comamonas sp., fibronectin type III modul, substrate-binding site.

scholarly journals Sequence, Structure, and Binding Analysis of Cyclodextrinase (TK1770) fromT. kodakarensis(KOD1) Using anIn SilicoApproach

Archaea ◽  
2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Ramzan Ali ◽  
Muhammad Imtiaz Shafiq
Keyword(s):  
Catalytic Domain ◽  
Substrate Binding ◽  
Homology Model ◽  
Catalytic Triad ◽  
Catalytic Site ◽  
Substrate Complex ◽  
Helix Loop Helix ◽  
Loop Region ◽  
Enzyme Substrate ◽  
Hydrolyzing Enzymes

Thermostable cyclodextrinase (Tk1770 CDase) from hyperthermophilic archaeonThermococcus kodakarensis(KOD1) hydrolyzes cyclodextrins into linear dextrins. The sequence of Tk1770 CDase retrieved from UniProt was aligned with sequences of sixteen CD hydrolyzing enzymes and a phylogenetic tree was constructed using Bayesian inference. The homology model of Tk1770 CDase was constructed and optimized with Modeller v9.14 program. The model was validated with ProSA server and PROCHECK analysis. Four conserved regions and the catalytic triad consisting of Asp411, Glu437, and Asp502 of GH13 family were identified in catalytic site. Also an additional fifth conserved region downstream to the fourth region was also identified. The structure of Tk1770 CDase consists of an additional N′-domain and a helix-loop-helix motif that is conserved in all archaeal CD hydrolyzing enzymes. The N′-domain contains an extended loop region that forms a part of catalytic domain and plays an important role in stability and substrate binding. The docking of substrate into catalytic site revealed the interactions with different conserved residues involved in substrate binding and formation of enzyme-substrate complex.

Characterization of Recombinant Human Coagulation Factor XFriuli

1996 ◽  
Vol 75 (02) ◽  
pp. 313-317 ◽  
Author(s):  
D J Kim ◽  
A Girolami ◽  
H L James
Keyword(s):  
Catalytic Domain ◽  
Coagulation Factor ◽  
Molecular Size ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Bleeding Tendency ◽  
Factor X ◽  
Amino Terminal ◽  
Normal Plasma ◽  
Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

scholarly journals Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yufei Han ◽  
Qian Zhuang ◽  
Bo Sun ◽  
Wenping Lv ◽  
Sheng Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Biochemical Characterization ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Transmembrane Segments ◽  
Therapeutic Molecules ◽  
Binding Cavity ◽  
Immune System Regulation ◽  
Mediated Reduction

AbstractSteroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

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