Selectivity of the surface binding site (SBS) on barley starch synthase I

Biologia ◽  
2014 ◽  
Vol 69 (9) ◽  
Author(s):  
Casper Wilkens ◽  
Jose Cuesta-Seijo ◽  
Monica Palcic ◽  
Birte Svensson
Keyword(s):  
Crystal Structure ◽  
Surface Plasmon Resonance ◽  
Binding Site ◽  
Active Site ◽  
Mutational Analysis ◽  
Starch Synthase ◽  
Surface Binding ◽  
Binding Studies ◽  
A Chain ◽  
Surface Binding Site

AbstractStarch synthase I (SSI) from various sources has been shown to preferentially elongate branch chains of degree of polymerisation (DP) from 6–7 to produce chains of DP 8–12. In the recently determined crystal structure of barley starch synthase I (HvSSI) a so-called surface binding site (SBS) was seen, which was found by mutational analysis to be essential for the activity of HvSSI on glycogen. We now show in binding studies using surface plasmon resonance that HvSSI has no detectable affinity for malto-triose and -tetraose, but clearly binds maltopentaose, -hexaose, -heptaose (M7) and β-cyclodextrin (β-CD) albeit with a measurable K D for only β-CD and M7. Moreover, an HvSSI SBS mutant F538A lost the ability to bind β-CD and maltooligosaccharides. This behaviour suggests that a chain in the α-glucan molecule (amylopectin) that is undergoing extension attaches itself at the SBS and that the active site itself, likely working on a different end chain, has low affinity for both substrate and product.

scholarly journals Crystal structure of a pyridoxal 5′-phosphate-dependent aspartate racemase derived from the bivalve mollusc Scapharca broughtonii

2017 ◽  
Vol 73 (12) ◽  
pp. 651-656 ◽  
Author(s):  
Taichi Mizobuchi ◽  
Risako Nonaka ◽  
Motoki Yoshimura ◽  
Katsumasa Abe ◽  
Shouji Takahashi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Active Site ◽  
Metal Binding ◽  
Bivalve Mollusc ◽  
Active Site Residues ◽  
X Ray ◽  
Aspartate Racemase ◽  
Scapharca Broughtonii

Aspartate racemase (AspR) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that is responsible for D-aspartate biosynthesis in vivo. To the best of our knowledge, this is the first study to report an X-ray crystal structure of a PLP-dependent AspR, which was resolved at 1.90 Å resolution. The AspR derived from the bivalve mollusc Scapharca broughtonii (SbAspR) is a type II PLP-dependent enzyme that is similar to serine racemase (SR) in that SbAspR catalyzes both racemization and dehydration. Structural comparison of SbAspR and SR shows a similar arrangement of the active-site residues and nucleotide-binding site, but a different orientation of the metal-binding site. Superposition of the structures of SbAspR and of rat SR bound to the inhibitor malonate reveals that Arg140 recognizes the β-carboxyl group of the substrate aspartate in SbAspR. It is hypothesized that the aromatic proline interaction between the domains, which favours the closed form of SbAspR, influences the arrangement of Arg140 at the active site.

scholarly journals The crystal structure of the Rv0301-Rv0300 VapBC-3 toxin-antitoxin complex from M. tuberculosis reveals a Mg2+ ion in the active site and a putative RNA-binding site

2012 ◽  
Vol 21 (11) ◽  
pp. 1754-1767 ◽  
Author(s):  
Andrew B. Min ◽  
Linda Miallau ◽  
Michael R. Sawaya ◽  
Jeff Habel ◽  
Duilio Cascio ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Active Site ◽  
Rna Binding

Fragment screening of inhibitors for MIF tautomerase reveals a cryptic surface binding site

2010 ◽  
Vol 20 (6) ◽  
pp. 1821-1824 ◽  
Author(s):  
Larry R. McLean ◽  
Ying Zhang ◽  
Hua Li ◽  
Yong-Mi Choi ◽  
Zuoning Han ◽  
...  
Keyword(s):  
Binding Site ◽  
Surface Binding ◽  
Fragment Screening ◽  
Surface Binding Site

scholarly journals The Importance of Surface-Binding Site towards Starch-Adsorptivity Level in α-Amylase: A Review on Structural Point of View

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Umi Baroroh ◽  
Muhammad Yusuf ◽  
Saadah Diana Rachman ◽  
Safri Ishmayana ◽  
Mas Rizky A. A. Syamsunarno ◽  
...  
Keyword(s):  
Binding Site ◽  
Large Scale ◽  
Point Of View ◽  
Carbohydrate Binding ◽  
Surface Binding ◽  
Raw Starch ◽  
Amylolytic Enzymes ◽  
Stacking Interaction ◽  
Structural Point ◽  
Surface Binding Site

Starch is a polymeric carbohydrate composed of glucose. As a source of energy, starch can be degraded by various amylolytic enzymes, including α-amylase. In a large-scale industry, starch processing cost is still expensive due to the requirement of high temperature during the gelatinization step. Therefore, α-amylase with raw starch digesting ability could decrease the energy cost by avoiding the high gelatinization temperature. It is known that the carbohydrate-binding module (CBM) and the surface-binding site (SBS) of α-amylase could facilitate the substrate binding to the enzyme’s active site to enhance the starch digestion. These sites are a noncatalytic module, which could interact with a lengthy substrate such as insoluble starch. The major interaction between these sites and the substrate is the CH/pi-stacking interaction with the glucose ring. Several mutation studies on the Halothermothrix orenii, SusG Bacteroides thetaiotamicron, Barley, Aspergillus niger, and Saccharomycopsis fibuligera α-amylases have revealed that the stacking interaction through the aromatic residues at the SBS is essential to the starch adsorption. In this review, the SBS in various α-amylases is also presented. Therefore, based on the structural point of view, SBS is suggested as an essential site in α-amylase to increase its catalytic activity, especially towards the insoluble starch.

scholarly journals Structural basis of HIV inhibition by translocation-defective RT inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA)

2016 ◽  
Vol 113 (33) ◽  
pp. 9274-9279 ◽  
Author(s):  
Zhe Li Salie ◽  
Karen A. Kirby ◽  
Eleftherios Michailidis ◽  
Bruno Marchand ◽  
Kamalendra Singh ◽  
...  
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Complex Structure ◽  
Primer Binding Site ◽  
Structural Basis ◽  
Inhibition Mechanism ◽  
Catalytic Complex ◽  
Hiv Reverse Transcriptase ◽  
A Chain ◽  
Ordered Water

4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is the most potent nucleoside analog inhibitor of HIV reverse transcriptase (RT). It retains a 3′-OH yet acts as a chain-terminating agent by diminishing translocation from the pretranslocation nucleotide-binding site (N site) to the posttranslocation primer-binding site (P site). Also, facile misincorporation of EFdA-monophosphate (MP) results in difficult-to-extend mismatched primers. To understand the high potency and unusual inhibition mechanism of EFdA, we solved RT crystal structures (resolutions from 2.4 to 2.9 Å) that include inhibition intermediates (i) before inhibitor incorporation (catalytic complex, RT/DNA/EFdA-triphosphate), (ii) after incorporation of EFdA-MP followed by dT-MP (RT/DNAEFdA-MPP•dT-MPN), or (iii) after incorporation of two EFdA-MPs (RT/DNAEFdA-MPP•EFdA-MPN); (iv) the latter was also solved with EFdA-MP mismatched at the N site (RT/DNAEFdA-MPP•EFdA-MP*N). We report that the inhibition mechanism and potency of EFdA stem from interactions of its 4′-ethynyl at a previously unexploited conserved hydrophobic pocket in the polymerase active site. The high resolution of the catalytic complex structure revealed a network of ordered water molecules at the polymerase active site that stabilize enzyme interactions with nucleotide and DNA substrates. Finally, decreased translocation results from favorable interactions of primer-terminating EFdA-MP at the pretranslocation site and unfavorable posttranslocation interactions that lead to observed localized primer distortions.

scholarly journals Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis

2011 ◽  
Vol 77 (16) ◽  
pp. 5730-5738 ◽  
Author(s):  
Hanna M. Dudek ◽  
Gonzalo de Gonzalo ◽  
Daniel E. Torres Pazmiño ◽  
Piotr Stępniak ◽  
Lucjan S. Wyrwicz ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Binding Site ◽  
Active Site ◽  
Structural Model ◽  
Mutational Analysis ◽  
Substrate Binding ◽  
Thermobifida Fusca ◽  
Content Type ◽  
Substrate Binding Site

ABSTRACTBaeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) fromThermobifida fuscais the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.

scholarly journals Identification of the site of oxidase substrate binding inScytalidium thermophilumcatalase

2018 ◽  
Vol 74 (10) ◽  
pp. 979-985 ◽  
Author(s):  
Yonca Yuzugullu Karakus ◽  
Gunce Goc ◽  
Sinem Balci ◽  
Briony A. Yorke ◽  
Chi H. Trinh ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Kinetic Analysis ◽  
Binding Site ◽  
Active Site ◽  
Oxidase Activity ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Scytalidium Thermophilum ◽  
Lateral Channel

The catalase fromScytalidium thermophilumis a homotetramer containing a hemedin each active site. Although the enzyme has a classical monofunctional catalase fold, it also possesses oxidase activity towards a number of small organics, including catechol and phenol. In order to further investigate this, the crystal structure of the complex of the catalase with the classical catalase inhibitor 3-amino-1,2,4-triazole (3TR) was determined at 1.95 Å resolution. Surprisingly, no binding to the heme site was observed; instead, 3TR occupies a binding site corresponding to the NADPH-binding pocket in mammalian catalases at the entrance to a lateral channel leading to the heme. Kinetic analysis of site-directed mutants supports the assignment of this pocket as the binding site for oxidase substrates.

scholarly journals A Novel Adenylate Binding Site Confers Phosphopantetheine Adenylyltransferase Interactions with Coenzyme A

2003 ◽  
Vol 185 (14) ◽  
pp. 4074-4080 ◽  
Author(s):  
Tina Izard
Keyword(s):  
Crystal Structure ◽  
High Resolution ◽  
Binding Site ◽  
Active Site ◽  
Binding Sites ◽  
Biosynthetic Pathway ◽  
Coenzyme A ◽  
Asymmetric Unit ◽  
Phosphate Moiety ◽  
Reversible Transfer

ABSTRACT Phosphopantetheine adenylyltransferase (PPAT) regulates the key penultimate step in the essential coenzyme A (CoA) biosynthetic pathway. PPAT catalyzes the reversible transfer of an adenylyl group from Mg2+:ATP to 4′-phosphopantetheine to form 3′-dephospho-CoA (dPCoA) and pyrophosphate. The high-resolution crystal structure of PPAT complexed with CoA has been determined. Remarkably, CoA and the product dPCoA bind to the active site in distinct ways. Although the phosphate moiety within the phosphopantetheine arm overlaps, the pantetheine arm binds to the same pocket in two distinct conformations, and the adenylyl moieties of these two ligands have distinct binding sites. Moreover, the PPAT:CoA crystal structure confirms the asymmetry of binding to the two trimers within the hexameric enzyme. Specifically, the pantetheine arm of CoA bound to one protomer within the asymmetric unit displays the dPCoA-like conformation with the adenylyl moiety disordered, whereas CoA binds the twofold-related protomer in an ordered and unique fashion.

scholarly journals Asp271 is critical for substrate interaction with the surface binding site in β-agarase a from Zobellia galactanivorans

2018 ◽  
Vol 87 (1) ◽  
pp. 34-40
Author(s):  
Casper Wilkens ◽  
Manish K. Tiwari ◽  
Helen Webb ◽  
Murielle Jam ◽  
Mirjam Czjzek ◽  
...  
Keyword(s):  
Binding Site ◽  
Surface Binding ◽  
Substrate Interaction ◽  
Surface Binding Site
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