scholarly journals The Structural Motifs for Substrate Binding and Dimerization of the α Subunit of Collagen Prolyl 4-Hydroxylase

Structure ◽  
2013 ◽  
Vol 21 (12) ◽  
pp. 2107-2118 ◽  
Author(s):  
Jothi Anantharajan ◽  
M. Kristian Koski ◽  
Petri Kursula ◽  
Reija Hieta ◽  
Ulrich Bergmann ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Structural Motifs ◽  
Α Subunit

Lysine164α of protein farnesyltransferase is important for both CaaX substrate binding and catalysis

2001 ◽  
Vol 360 (3) ◽  
pp. 625-631 ◽  
Author(s):  
Kendra E. HIGHTOWER ◽  
Smita DE ◽  
Carolyn WEINBAUM ◽  
Rebecca A. SPENCE ◽  
Patrick J. CASEY
Keyword(s):  
Rate Constant ◽  
Positive Charge ◽  
Substrate Binding ◽  
Product Formation ◽  
Chemical Mechanism ◽  
Α Subunit ◽  
Protein Farnesyltransferase ◽  
Oncogenic Ras ◽  
Tumour Formation ◽  
Mechanism Of Catalysis

Protein farnesyltransferase (FTase) catalyses the formation of a thioether linkage between proteins containing a C-terminal CaaX motif and a 15-carbon isoprenoid. The involvement of substrates such as oncogenic Ras proteins in tumour formation has led to intense efforts in targeting this enzyme for development of therapeutics. In an ongoing programme to elucidate the mechanism of catalysis by FTase, specific residues of the enzyme identified in structural studies as potentially important in substrate binding and catalysis are being targeted for mutagenesis. In the present study, the role of the positive charge of Lys164 of the α subunit of FTase in substrate binding and catalysis was investigated. Comparison of the wild-type enzyme with enzymes that have either an arginine or alanine residue substituted at this position revealed unexpected roles for this residue in both substrate binding and catalysis. Removal of the positive charge had a significant effect on the association rate constant and the binding affinity of a CaaX peptide substrate, indicating that the positive charge of Lys164α is involved in formation of the enzyme (E)·farnesyl diphosphate (FPP)·peptide ternary complex. Furthermore, mutation of Lys164α resulted in a substantial decrease in the observed rate constant for product formation without alteration of the chemical mechanism. These and additional studies provide compelling evidence that both the charge on Lys164α, as well as the positioning of the charge, are important for overall catalysis by FTase.

scholarly journals Assessment of Toluene/Biphenyl Dioxygenase Gene Diversity in Benzene-Polluted Soils: Links between Benzene Biodegradation and Genes Similar to Those Encoding Isopropylbenzene Dioxygenases

2006 ◽  
Vol 72 (5) ◽  
pp. 3504-3514 ◽  
Author(s):  
Robert Witzig ◽  
Howard Junca ◽  
Hans-J�rgen Hecht ◽  
Dietmar H. Pieper
Keyword(s):  
Pseudomonas Fluorescens ◽  
Gene Diversity ◽  
Substrate Binding ◽  
Single Strand Conformation Polymorphism ◽  
Binding Pocket ◽  
Bacterial Isolates ◽  
Substrate Binding Pocket ◽  
Soil Dna ◽  
Α Subunit ◽  
Substrate Utilization Pattern

ABSTRACT The PCR-single-strand conformation polymorphism (SSCP) technique was used to assess the diversity and distribution of Rieske nonheme iron oxygenases of the toluene/biphenyl subfamily in soil DNA and bacterial isolates recovered from sites contaminated with benzene, toluene, ethylbenzene, and xylenes (BTEX). The central cores of genes encoding the catalytic α subunits were targeted, since they are responsible for the substrate specificities of these enzymes. SSCP functional genotype fingerprinting revealed a substantial diversity of oxygenase genes in three differently BTEX-contaminated soil samples, and sequence analysis indicated that in both the soil DNA and the bacterial isolates, genes for oxygenases related to the isopropylbenzene (cumene) dioxygenase branch of the toluene/biphenyl oxygenase subfamily were predominant among the detectable genotypes. The peptide sequences of the two most abundant α subunit sequence types differed by only five amino acids (residues 258, 286, 288, 289, and 321 according to numbering in cumene dioxygenase α subunit CumA1 of Pseudomonas fluorescens IP01). However, a strong correlation between sequence type and substrate utilization pattern was observed in isolates harboring these genes. Two of these residues were located at positions contributing, according to the resolved crystal structure of cumene dioxygenase from Pseudomonas fluorescens IP01, to the inner surface of the substrate-binding pocket. Isolates containing an α subunit with isoleucine and leucine at positions 288 and 321, respectively, were capable of degrading benzene and toluene, whereas isolates containing two methionine substitutions were found to be incapable of degrading toluene, indicating that the more bulky methionine residues significantly narrowed the available space within the substrate-binding pocket.

scholarly journals Exploring structural motifs necessary for substrate binding in the active site of Escherichia coli pantothenate kinase

2014 ◽  
Vol 22 (12) ◽  
pp. 3083-3090 ◽  
Author(s):  
Emelia Awuah ◽  
Eric Ma ◽  
Annabelle Hoegl ◽  
Kenward Vong ◽  
Eric Habib ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Substrate Binding ◽  
Pantothenate Kinase ◽  
Structural Motifs

scholarly journals Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Cristina Olivieri ◽  
Caitlin Walker ◽  
Adak Karamafrooz ◽  
Yingjie Wang ◽  
V. S. Manu ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Molecular Mechanisms ◽  
Substrate Binding ◽  
Chimeric Protein ◽  
Α Subunit ◽  
Primary Driver ◽  
Chimeric Construct ◽  
J Domain ◽  
Kinase A

AbstractAn aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-CDNAJB1) in which the J-domain of the heat shock protein 40 is fused to the catalytic α subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-CDNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera’s allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-CDNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.

scholarly journals Structural basis for substrate recognition mechanism of ER glucosidase II

2014 ◽  
Vol 70 (a1) ◽  
pp. C300-C300
Author(s):  
Tadashi Satoh ◽  
Takayasu Toshimori ◽  
Takumi Yamaguchi ◽  
Zhu Tong ◽  
Koichi Kato
Keyword(s):  
Crystal Structure ◽  
Quality Control ◽  
Control System ◽  
Substrate Binding ◽  
Terminal Segment ◽  
Glycoside Hydrolases ◽  
Quality Control System ◽  
Structural Basis ◽  
Α Subunit ◽  
Glucosidase Ii

The endoplasmic reticulum (ER) possesses a sophisticated quality control system to proofread newly synthesized proteins. A series of N-linked oligosaccharide intermediates attached on the nascent proteins serves as specific tags for the quality control system. In this system, glucosidase II is involved in trimming of non-reducing terminal glucose residue of N-glycan intermediates. Glucosidase II consists of approximately 110 kDa catalytic α subunit (GIIα) and 60 kDa non-catalytic regulatory β subunit (GIIβ). It has been shown that GIIα alone can hydrolyze a small α-glycosidase model substrate (pNP-glucose), while it cannot catalyze deglucosylation of the N-linked oligosaccharide substrates unless it makes a complex with GIIβ. In this study, we determined the first crystal structure of GIIα in the absence and presence of its inhibitor 1-deoxynojirimycin at 1.6-Å resolution. The crystal structure revealed that GIIα has a characteristic segment at the N-terminus as compared with the cognate glycoside hydrolases (GH31). Interestingly, the N-terminal segment was accommodated on the substrate-binding pocket. Based on these results, we suggest that the N-terminal segment of GIIα undergoes structural rearrangement through interaction with GIIβ, thereby promoting the substrate-binding capacity for the N-linked oligosaccharide substrates.

scholarly journals Crystal Structure of Adenylylsulfate Reductase from Desulfovibrio gigas Suggests a Potential Self-Regulation Mechanism Involving the C Terminus of the β-Subunit

2009 ◽  
Vol 191 (24) ◽  
pp. 7597-7608 ◽  
Author(s):  
Yuan-Lan Chiang ◽  
Yin-Cheng Hsieh ◽  
Jou-Yin Fang ◽  
En-Hong Liu ◽  
Yen-Chieh Huang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Active Site ◽  
Substrate Binding ◽  
Regulation Mechanism ◽  
Β Subunit ◽  
Α Subunit ◽  
Desulfovibrio Gigas ◽  
C Terminus ◽  
Terminal Loop

ABSTRACT Adenylylsulfate reductase (adenosine 5′-phosphosulfate [APS] reductase [APSR]) plays a key role in catalyzing APS to sulfite in dissimilatory sulfate reduction. Here, we report the crystal structure of APSR from Desulfovibrio gigas at 3.1-Å resolution. Different from the α2β2-heterotetramer of the Archaeoglobus fulgidus, the overall structure of APSR from D. gigas comprises six αβ-heterodimers that form a hexameric structure. The flavin adenine dinucleotide is noncovalently attached to the α-subunit, and two [4Fe-4S] clusters are enveloped by cluster-binding motifs. The substrate-binding channel in D. gigas is wider than that in A. fulgidus because of shifts in the loop (amino acid 326 to 332) and the α-helix (amino acid 289 to 299) in the α-subunit. The positively charged residue Arg160 in the structure of D. gigas likely replaces the role of Arg83 in that of A. fulgidus for the recognition of substrates. The C-terminal segment of the β-subunit wraps around the α-subunit to form a functional unit, with the C-terminal loop inserted into the active-site channel of the α-subunit from another αβ-heterodimer. Electrostatic interactions between the substrate-binding residue Arg282 in the α-subunit and Asp159 in the C terminus of the β-subunit affect the binding of the substrate. Alignment of APSR sequences from D. gigas and A. fulgidus shows the largest differences toward the C termini of the β-subunits, and structural comparison reveals notable differences at the C termini, activity sites, and other regions. The disulfide comprising Cys156 to Cys162 stabilizes the C-terminal loop of the β-subunit and is crucial for oligomerization. Dynamic light scattering and ultracentrifugation measurements reveal multiple forms of APSR upon the addition of AMP, indicating that AMP binding dissociates the inactive hexamer into functional dimers, presumably by switching the C terminus of the β-subunit away from the active site. The crystal structure of APSR, together with its oligomerization properties, suggests that APSR from sulfate-reducing bacteria might self-regulate its activity through the C terminus of the β-subunit.

Fibronexus-Like Adhesion Sites are Present at the Invasive Zones of Human Epidermoid Carcinomas

1982 ◽  
Vol 40 ◽  
pp. 106-109
Author(s):  
Irwin I. Singer
Keyword(s):  
Cell Cycle ◽  
Serum Concentration ◽  
Substrate Binding ◽  
High Serum ◽  
Adhesion Sites ◽  
Substrate Adhesion ◽  
Focal Contacts ◽  
Adhesion Complex ◽  
Low Serum

Our previous results indicate that two types of fibronectin-cytoskeletal associations may be formed at the fibroblast surface: dorsal matrixbinding fibronexuses generated in high serum (5% FBS) cultures, and ventral substrate-adhering units formed in low serum (0.3% FBS) cultures. The substrate-adhering fibronexus consists of at least vinculin (VN) and actin in its cytoplasmic leg, and fibronectin (FN) as one of its major extracellular components. This substrate-adhesion complex is localized in focal contacts, the sites of closest substratum approach visualized with interference reflection microscopy, which appear to be the major points of cell-tosubstrate adhesion. In fibroblasts, the latter substrate-binding complex is characteristic of cultures that are arrested at the G1 phase of the cell cycle due to the low serum concentration in their medium. These arrested fibroblasts are very well spread, flattened, and immobile.

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