Probing a novel potato lipoxygenase with dual positional specificity reveals primary determinants of substrate binding and requirements for a surface hydrophobic loop and has implications for the role of lipoxygenases in tubers

2001 ◽  
Vol 353 (2) ◽  
pp. 345-355 ◽  
Author(s):  
Richard K. HUGHES ◽  
Stuart I. WEST ◽  
Andrzej R. HORNOSTAJ ◽  
David M. LAWSON ◽  
Shirley A. FAIRHURST ◽  
...  
Keyword(s):  
Potato Tuber ◽  
Critical Role ◽  
Homology Model ◽  
Site Directed Mutagenesis ◽  
Eicosatetraenoic Acid ◽  
Positional Specificity ◽  
Binding Pockets ◽  
Plant Lipoxygenases ◽  
A Minor

A new potato tuber lipoxygenase full-length cDNA sequence (lox1:St:2) has been isolated from potato tubers and used to express in Escherichia coli and characterize a novel recombinant lipoxygenase (potato 13/9-lipoxygenase). Like most plant lipoxygenases it produced carbonyl compounds from linoleate (the preferred substrate) and was purified in the Fe(II) (ferrous) state. Typical of other potato tuber lipoxygenases, it produced 5-HPETE [5(S)-hydroperoxy-(6E, 8Z, 11Z, 14Z)-eicosatetraenoic acid] from arachidonate. In contrast to any other potato tuber lipoxygenase, it exhibited dual positional specificity and produced roughly equimolar amounts of 13- and 9-hydroperoxides (or only a slight molar excess of 9-hydroperoxides) from linoleate. We have used a homology model of pea 9/13-lipoxygenase to superimpose and compare the linoleate-binding pockets of different potato lipoxygenases of known positional specificity. We then tested this model by using site-directed mutagenesis to identify some primary determinants of linoleate binding to potato 13/9-lipoxygenase and concluded that the mechanism determining positional specificity described for a cucumber lipoxygenase does not apply to potato 13/9-lipoxygenase. This supports our previous studies on pea seed lipoxygenases for the role of pocket volume rather than inverse orientation as a determinant of dual positional specificity in plant lipoxygenases. We have also used deletion mutagenesis to identify a critical role in catalysis for a surface hydrophobic loop in potato 13/9-lipoxygenase and speculate that this may control substrate access. Although potato 13/9-lipoxygenase represents only a minor isoform in tubers, such evidence for a single lipoxygenase species with dual positional specificity in tubers has implications for the proposed role of potato lipoxygenases in the plant.

Access of the substrate to the active site of squalene and oxidosqualene cyclases: comparative inhibition, site-directed mutagenesis and homology-modelling studies

2005 ◽  
Vol 33 (5) ◽  
pp. 1202-1205 ◽  
Author(s):  
S. Oliaro-Bosso ◽  
T. Schulz-Gasch ◽  
S. Taramino ◽  
M. Scaldaferri ◽  
F. Viola ◽  
...  
Keyword(s):  
Active Site ◽  
Homology Modelling ◽  
Critical Role ◽  
Homology Model ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Specific Substrate ◽  
Oxidosqualene Cyclase ◽  
Active Site Cavity

Substrate access to the active-site cavity of squalene-hopene cyclase from Alicyclobacillus acidocaldarious and lanosterol synthase [OSC (oxidosqualene cyclase)] from Saccharomyces cerevisiae was studied by an inhibition, mutagenesis and homology-modelling approach. Crystal structure and homology modelling indicate that both enzymes possess a narrow constriction that separates an entrance lipophilic channel from the active-site cavity. The role of the constriction as a mobile gate that permits substrate passage was investigated by experiments in which critically located Cys residues, either present in native protein or inserted by site-directed mutagenesis, were labelled with specifically designed thiol-reacting molecules. Some amino acid residues of the yeast enzyme, selected on the basis of sequence alignment and a homology model, were individually replaced by residues bearing side chains of different lengths, charges or hydrophobicities. In some of these mutants, substitution severely reduced enzymatic activity and thermal stability. Homology modelling revealed that in these mutants some critical stabilizing interactions could no longer occur. The possible critical role of entrance channel and constriction in specific substrate recognition by eukaryotic OSC is discussed.

scholarly journals Crystal structure of the family 7 endoglucanase I (Cel7B) from Humicola insolens at 2.2 Å resolution and identification of the catalytic nucleophile by trapping of the covalent glycosyl-enzyme intermediate

1998 ◽  
Vol 335 (2) ◽  
pp. 409-416 ◽  
Author(s):  
Lloyd F. MACKENZIE ◽  
Gerlind SULZENBACHER ◽  
Christina DIVNE ◽  
T. Alwyn JONES ◽  
Helle F. WÖLDIKE ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Critical Role ◽  
Soft Rot ◽  
Site Directed Mutagenesis ◽  
Cellobiohydrolase I ◽  
Native Form ◽  
Humicola Insolens ◽  
Endoglucanase I ◽  
Enzyme Intermediate

Cellulose is the major polysaccharide component of the plant cell wall and the most abundant naturally produced macromolecule on Earth. The enzymic degradation of cellulose, by cellulases, is therefore of great environmental and commercial significance. Cellulases are found in 12 of the glycoside hydrolase families classified according to their amino acid sequence similarities. Endoglucanase I (Cel7B), from the soft-rot fungus Humicola insolens, is a family 7 enzyme. The structure of the native form of Cel7B from H. insolens at 2.2 Å resolution has been solved by molecular replacement using the known Trichoderma reesei cellobiohydrolase I [Divne, Ståhlberg, Reinikainen, Ruohonen, Pettersson, Knowles, Teeri and Jones (1994) Science265, 524–528] structure as the search model. Cel7B catalyses hydrolysis of the β-1,4 glycosidic linkages in cellulose with net retention of anomeric configuration. The catalytic nucleophile at the active site of Cel7B has been identified as Glu-197 by trapping of a 2-deoxy-2-fluorocellotriosyl enzyme intermediate and identification of the labelled peptide in peptic digests by tandem MS. Site-directed mutagenesis of both Glu-197 and the prospective catalytic acid, Glu-202, results in inactive enzyme, confirming the critical role of these groups for catalysis.

scholarly journals Albino mutants of Streptomyces glaucescens tyrosinase

1991 ◽  
Vol 274 (3) ◽  
pp. 707-713 ◽  
Author(s):  
M P Jackman ◽  
A Hajnal ◽  
K Lerch
Keyword(s):  
Active Site ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Mutant Proteins ◽  
Carbonyl Derivatives ◽  
Hydrogen Bonding Interactions ◽  
Albino Phenotype ◽  
Derivatives Of ◽  
Streptomyces Glaucescens

Site-directed mutagenesis was used to determine the functional role of several residues of Streptomyces glaucescens tyrosinase. Replacement of His-37, -53, -193 or -215 by glutamine yields albino phenotypes, as determined by expression on melanin-indicator plates. The purified mutant proteins display no detectable oxy-enzyme and increased Cu lability at the binuclear active site. The carbonyl derivatives of H189Q and H193Q luminesce, with lambda max. displaced more than 25 nm to a longer wavelength compared with native tyrosinase. The remaining histidine mutants display no detectable luminescence. The results are consistent with these histidine residues (together with His-62 and His-189 reported earlier) acting as Cu ligands in the Streptomyces glaucescens enzyme. Conservative substitution of the invariant Asn-190 by glutamine also gives an albino phenotype, no detectable oxy-enzyme and labilization of active-site Cu. The luminescence spectrum of carbonyl-N190Q, however, closely resembles that of the native enzyme under conditions promoting double Cu occupancy of the catalytic site. A critical role for Asn-190 in active-site hydrogen-bonding interactions is proposed.

Phosphorylation of Ser136 is critical for potent bone sialoprotein-mediated nucleation of hydroxyapatite crystals

2010 ◽  
Vol 428 (3) ◽  
pp. 385-395 ◽  
Author(s):  
Gurpreet S. Baht ◽  
Jason O'Young ◽  
Antonia Borovina ◽  
Hong Chen ◽  
Coralee E. Tye ◽  
...  
Keyword(s):  
Critical Role ◽  
Bone Sialoprotein ◽  
Site Directed Mutagenesis ◽  
Crystal Face ◽  
Mineralized Tissues ◽  
Dynamics Simulations ◽  
A Site ◽  
Functional Analyses ◽  
Kinase Ck2

Acidic phosphoproteins of mineralized tissues such as bone and dentin are believed to play important roles in HA (hydroxyapatite) nucleation and growth. BSP (bone sialoprotein) is the most potent known nucleator of HA, an activity that is thought to be dependent on phosphorylation of the protein. The present study identifies the role phosphate groups play in mineral formation. Recombinant BSP and peptides corresponding to residues 1–100 and 133–205 of the rat sequence were phosphorylated with CK2 (protein kinase CK2). Phosphorylation increased the nucleating activity of BSP and BSP-(133–205), but not BSP-(1–100). MS analysis revealed that the major site phosphorylated within BSP-(133–205) was Ser136, a site adjacent to the series of contiguous glutamate residues previously implicated in HA nucleation. The critical role of phosphorylated Ser136 in HA nucleation was confirmed by site-directed mutagenesis and functional analyses. Furthermore, peptides corresponding to the 133–148 sequence of rat BSP were synthesized with or without a phosphate group on Ser136. As expected, the phosphopeptide was a more potent nucleator. The mechanism of nucleation was investigated using molecular-dynamics simulations analysing BSP-(133–148) interacting with the {100} crystal face of HA. Both phosphorylated and non-phosphorylated sequences adsorbed to HA in extended conformations with alternating residues in contact with and facing away from the crystal face. However, this alternating-residue pattern was more pronounced when Ser136 was phosphorylated. These studies demonstrate a critical role for Ser136 phosphorylation in BSP-mediated HA nucleation and identify a unique mode of interaction between the nucleating site of the protein and the {100} face of HA.

scholarly journals A Critical Role of Non-active Site Residues on Cyclooxygenase Helices 5 and 6 in the Control of Prostaglandin Stereochemistry at Carbon 15

2007 ◽  
Vol 282 (38) ◽  
pp. 28157-28163 ◽  
Author(s):  
Karin Valmsen ◽  
William E. Boeglin ◽  
Reet Järving ◽  
Ivar Järving ◽  
Külliki Varvas ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Amino Acid Sequence Identity ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Active Site Residues ◽  
The Core ◽  
The Difference

The correct stereochemistry of prostaglandins is a prerequisite of their biological activity and thus is under a strict enzymatic control. Recently, we cloned and characterized two cyclooxygenase (COX) isoforms in the coral Plexaura homomalla that share 97% amino acid sequence identity, yet form prostaglandins with opposite stereochemistry at carbon 15. The difference in oxygenation specificity is only partially accounted for by the single amino acid substitution in the active site (Ile or Val at position 349). For further elucidation of residues involved in the C-15 stereocontrol, a series of sequence swapping and site-directed mutagenesis experiments between 15R- and 15S-COX were performed. Our results show that the change in stereochemistry at carbon 15 of prostaglandins relates mainly to five amino acid substitutions on helices 5 and 6 of the coral COX. In COX proteins, these helices form a helix-turn-helix motif that traverses through the entire protein, contributing to the second shell of residues around the oxygenase active site; it constitutes the most highly conserved region where even slight changes result in loss of catalytic activity. The finding that this region is among the least conserved between the P. homomalla 15S- and 15R-specific COX further supports its significance in maintaining the desired prostaglandin stereochemistry at C-15. The results are particularly remarkable because, based on its strong conservation, the conserved middle of helix 5 is considered as central to the core structure of peroxidases, of which COX proteins are derivatives. Now we show that the same parts of the protein are involved in the control of oxygenation with 15R or 15S stereospecificity in the dioxygenase active site.

scholarly journals Critical Role of Cys168 in Noggin Protein's Biological Function

2005 ◽  
Vol 37 (3) ◽  
pp. 181-185
Author(s):  
Wei-Dong Liu ◽  
Xiang-Ling Feng ◽  
Cai-Ping Ren ◽  
Jian-Ling Shi ◽  
Xu-Yu Yang ◽  
...  
Keyword(s):  
Biological Activities ◽  
Critical Role ◽  
Ectopic Expression ◽  
Disulfide Bridge ◽  
Site Directed Mutagenesis ◽  
Morphogenetic Protein ◽  
Neural Cell Adhesion ◽  
A Site ◽  
The Relationship

Abstract Previous that noggin exerts its neural inducing effect by binding and antagonizing bone morphogenetic protein 4 (BMP4). In order to further clarify the relationship between the structure and the function of noggin, and elucidate the possible mechanism responsible for noggin-BMP4 interaction, we generated three noggin mutants, C168S, C174S and C197S, by using a site-directed mutagenesis method. Ectopic expression of wild-type (WT) noggin, C174S or C197S, in Xenopus animal caps (ACs) by mRNA injection converted the explants (prospective ectoderm) into neural tissue, as indicated by the neural-like morphology and expression of the neural cell adhesion molecule (NCAM) in the ACs. In contrast, ACs expressing C168S suffered an epidermal fate similar to the control caps. Similarly, among the three mutants, only C168S lost the dorsalizing function. These studies highlight the critical role played by Cys168 in noggin's biological activities. It probably participates in the formation of an intermolecular disulfide bridge.

Effect of matrix metallopeptidase 13 (MMP13) on multiple myeloma (MM) cells, osteoclast (OCL) activity, and bone resorption.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 8099-8099
Author(s):  
Jing Fu ◽  
Shirong Li ◽  
Rentian Feng ◽  
Mei Hua Jin ◽  
Farideh Sabeh ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Bone Resorption ◽  
Bone Disease ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Bone Lesions ◽  
Lytic Lesions

8099 Background: MM cells produce OCL-activating factors that induce excessive bone resorption resulting in lytic lesions. The role of MMPs in invasion/progression of solid tumors is well-known, but its function in MM has not been well elucidated. Our group has shown that MMP13 is highly expressed in primary MM cells and in sera of MM patients. Levels of MMP13 significantly correlate with the extent of bone disease. MMP13 is induced by IL-6 via AP-1 activation in MM cells and enhances fusion of OCL precursors resulting in excessive bone resorption. OCL formation using MNCs of mmp-13-/- mice resulted in a fusion defect, significantly decreased OCL size and activity, which could be reversed by exogenous MMP13 (ASH 2009, IMW 2011). Methods: Methods will be presented in the Results section. Results: RT-PCR and western blotting revealed that IL-6 treatment of MM cells induced MMP13 transcription (30-fold) and secretion (>1000-fold). Protein expression of the AP-1 members c-Jun and c-Fos was induced by IL-6, which correlated with MMP13 upregulation. Our data further indicate that the catalytic activity of MMP13 is not required to enhance OCL formation and bone resorption. To prove this, we generated the MMP13 activity-dead mutation MMP13-E223A construct by site-directed mutagenesis PCR-based cloning. The mutated protein was overexpressed in HEK293 cells and purified from the supernatant to confirm whether loss of catalytic activity blocks MMP13 function. To further investigate the in vivo role of MMP13 in MM bone disease, MMP13 expression was knocked down (KD) in murine 5TGM1-MM cells by pKLO. 1 puro lentiviral infection containing sh-RNA targeting mouse MMP13 sequence. MMP13-KD 5TGM1-MM cells or WT-5TGM1-MM cells were intratibially injected into RAG2-/- mice. Development of lytic bone lesions are monitored by micro-QCT and data will be available at the time of presentation. Conclusions: Our data suggest that MMP13, secreted by MM cells, plays a critical role in the development of lytic lesions. Targeting MMP13 represents a promising approach to treat or to prevent bone disease in MM.

scholarly journals Gain-of-function ADAMTS13 variants that are resistant to autoantibodies against ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura

Blood ◽  
2012 ◽  
Vol 119 (16) ◽  
pp. 3836-3843 ◽  
Author(s):  
Cui Jian ◽  
Juan Xiao ◽  
Lingjie Gong ◽  
Christopher G. Skipwith ◽  
Sheng-Yu Jin ◽  
...  
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Specific Activity ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Gain Of Function ◽  
Thrombocytopenic Purpura ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Thrombotic thrombocytopenic purpura (TTP) is primarily caused by immunoglobulin G (IgG) autoantibodies against A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats, 13 (ADAMTS13). Nearly all adult idiopathic TTP patients harbor IgGs, which bind the spacer domain of ADAMTS13, a region critical for recognition and proteolysis of von Willebrand factor (VWF). We hypothesize that a modification of an exosite in the spacer domain may generate ADAMTS13 variants with reduced autoantibody binding while preserving or enhancing specific activity. Site-directed mutagenesis was used to generate a series of ADAMTS13 variants, and their functional properties were assessed. Of 24 novel ADAMTS13 variants, 2 (ie, M4, R660K/F592Y/R568K/Y661F and M5, R660K/F592Y/R568K/Y661F/Y665F) exhibited increased specific activity approximately 4- to 5-fold and approximately 10- to 12-fold cleaving a peptide VWF73 substrate and multimeric VWF, respectively. More interestingly, the gain-of-function ADAMTS13 variants were more resistant to inhibition by anti-ADAMTS13 autoantibodies from patients with acquired idiopathic TTP because of reduced binding by anti-ADAMTS13 IgGs. These results shed more light on the critical role of the exosite in the spacer domain in substrate recognition. Our findings also help understand the pathogenesis of acquired autoimmune TTP. The autoantibody-resistant ADAMTS13 variants may be further developed as a novel therapeutic for acquired TTP with inhibitors.

scholarly journals Host EPAC1 modulates rickettsial adhesion to vascular endothelial cells via regulation of ANXA2 Y23 phosphorylation

2021 ◽  
Author(s):  
Zhengchen Su ◽  
Thomas Shelite ◽  
Yuan Qiu ◽  
Qing Chang ◽  
Maki Wakamiya ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Vascular Endothelial Cells ◽  
Spotted Fever ◽  
Critical Role ◽  
Initial Step ◽  
Site Directed Mutagenesis ◽  
Intracellular Camp ◽  
Spotted Fever Group

AbstractRecently we have identified that endothelial surface annexin A2 (ANAX2) functions as a receptor for spotted fever group rickettsial adhesin outer membrane protein B (OmpB), which binds to the endothelial cell (EC) surface. Moreover, we reported that intracellular cAMP receptor EPAC1 modulates ANXA2 tyrosine (Y) 23 phosphorylation, and inactivation of EPAC1 suppresses ANXA2 expression on the EC luminal surface by downregulating Y23 phosphorylation. Since we reported that EPAC1 plays a critical role in the initial step to successfully establish rickettsial infection of ECs, this work aims to answer the following: (a) What is the mechanism underlying the regulatory role of EPAC1 in ECs during the initial step of bacterial infection? (b) Is the EPAC1-ANXA2 signaling pathway involved in the regulation of rickettsial adhesion to ECs?In the present study, an established system that is anatomically-based and quantifies bacterial adhesion to ECs in vivo was combined with novel fluidic force microscopy (FluidFM) to dissect the functional role of the EPAC1-ANXA2 signaling pathway in rickettsiae–EC adhesion. We reveal that the deletion of the EPAC1 gene impedes rickettsial binding to endothelium in vivo. In addition, single living brain microvascular EC study that employs FluidFM and site-directed mutagenesis provides evidence that supports our finding that EPAC1 governs rickettsial adhesion to EC surfaces via regulation of ANXA2 Y23 phosphorylation.

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