scholarly journals Shaping Substrate Selectivity in a Broad-Spectrum Metallo-β-Lactamase

2018 ◽  
Vol 62 (4) ◽  
Author(s):  
Lisandro J. González ◽  
Cintia Stival ◽  
Juan L. Puzzolo ◽  
Diego M. Moreno ◽  
Alejandro J. Vila
Keyword(s):  
Broad Spectrum ◽  
Active Site ◽  
Substrate Recognition ◽  
Chimeric Protein ◽  
Major Group ◽  
Substrate Selectivity ◽  
Incomplete Knowledge ◽  
Naturally Occurring ◽  
The One

ABSTRACTMetallo-β-lactamases (MBLs) are the major group of carbapenemases produced by bacterial pathogens. The design of MBL inhibitors has been limited by, among other issues, incomplete knowledge about how these enzymes modulate substrate recognition. While most MBLs are broad-spectrum enzymes, B2 MBLs are exclusive carbapenemases. This narrower substrate profile has been attributed to a sequence insertion present in B2 enzymes that limits accessibility to the active site. In this work, we evaluate the role of sequence insertions naturally occurring in the B2 enzyme Sfh-I and in the broad-spectrum B1 enzyme SPM-1. We engineered a chimeric protein in which the sequence insertion of SPM-1 was replaced by the one present in Sfh-I. The chimeric variant is a selective cephalosporinase, revealing that the substrate profile of MBLs can be further tuned depending on the protein context. These results also show that the stable scaffold of MBLs allows a modular engineering much richer than the one observed in nature.

Role of active site arginine residues in substrate recognition by PPM1A

2021 ◽  
Author(s):  
Itsumi Tani ◽  
Shogo Ito ◽  
Yukiko Shirahata ◽  
Yutaka Matsuyama ◽  
James G. Omichinski ◽  
...  
Keyword(s):  
Active Site ◽  
Substrate Recognition ◽  
Arginine Residues

Differential Contribution of Active Site Residues in Substrate Recognition Sites 1 and 5 to Cytochrome P450 2C8 Substrate Selectivity and Regioselectivity†

Biochemistry ◽  
2004 ◽  
Vol 43 (24) ◽  
pp. 7834-7842 ◽  
Author(s):  
Oranun Kerdpin ◽  
David J. Elliot ◽  
Sanford L. Boye ◽  
Donald J. Birkett ◽  
Krongtong Yoovathaworn ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Active Site ◽  
Substrate Recognition ◽  
Substrate Selectivity ◽  
Active Site Residues ◽  
Recognition Sites ◽  
Cytochrome P450 2C8 ◽  
Differential Contribution

scholarly journals New insights into the mechanism of substrates trafficking in Glyoxylate/Hydroxypyruvate reductases

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Louise Lassalle ◽  
Sylvain Engilberge ◽  
Dominique Madern ◽  
Pierre Vauclare ◽  
Bruno Franzetti ◽  
...  
Keyword(s):  
Renal Failure ◽  
Active Site ◽  
Ternary Complex ◽  
Substrate Recognition ◽  
Binding Mode ◽  
X Ray ◽  
Hyperoxaluria Type

Abstract Glyoxylate accumulation within cells is highly toxic. In humans, it is associated with hyperoxaluria type 2 (PH2) leading to renal failure. The glyoxylate content within cells is regulated by the NADPH/NADH dependent glyoxylate/hydroxypyruvate reductases (GRHPR). These are highly conserved enzymes with a dual activity as they are able to reduce glyoxylate to glycolate and to convert hydroxypyruvate into D-glycerate. Despite the determination of high-resolution X-ray structures, the substrate recognition mode of this class of enzymes remains unclear. We determined the structure at 2.0 Å resolution of a thermostable GRHPR from Archaea as a ternary complex in the presence of D-glycerate and NADPH. This shows a binding mode conserved between human and archeal enzymes. We also determined the first structure of GRHPR in presence of glyoxylate at 1.40 Å resolution. This revealed the pivotal role of Leu53 and Trp138 in substrate trafficking. These residues act as gatekeepers at the entrance of a tunnel connecting the active site to protein surface. Taken together, these results allowed us to propose a general model for GRHPR mode of action.

Role of Two Residues Proximal to the Active Site of Ubc9 in Substrate Recognition by the Ubc9·SUMO-1 Thiolester Complex†

Biochemistry ◽  
2003 ◽  
Vol 42 (11) ◽  
pp. 3168-3179 ◽  
Author(s):  
Michael H. Tatham ◽  
Yuan Chen ◽  
Ronald T. Hay
Keyword(s):  
Active Site ◽  
Substrate Recognition

scholarly journals Expression, purification, crystallization and preliminary X-ray diffraction analysis of acylpeptide hydrolase fromDeinococcus radiodurans

2014 ◽  
Vol 70 (9) ◽  
pp. 1292-1295 ◽  
Author(s):  
Venkata Narayana Are ◽  
Biplab Ghosh ◽  
Ashwani Kumar ◽  
Pooja Yadav ◽  
Deepak Bhatnagar ◽  
...  
Keyword(s):  
Active Site ◽  
Mammalian Cells ◽  
Deinococcus Radiodurans ◽  
Structural Information ◽  
Screening Method ◽  
Substrate Selectivity ◽  
Cell Parameters ◽  
Acylpeptide Hydrolase ◽  
Monomeric Structure

Acylpeptide hydrolase (APH; EC 3.4.19.1), which belongs to the S9 family of serine peptidases (MEROPS), catalyzes the removal of anN-acylated amino acid from a blocked peptide. The role of this enzyme in mammalian cells has been suggested to be in the clearance of oxidatively damaged proteins as well as in the degradation of the β-amyloid peptides implicated in Alzheimer's disease. Detailed structural information for the enzyme has been reported from two thermophilic archaea; both of the archaeal APHs share a similar monomeric structure. However, the mechanisms of substrate selectivity and active-site accessibility are totally different and are determined by inter-domain flexibility or the oligomeric structure. An APH homologue from a bacterium,Deinococcus radiodurans(APHdr), has been crystallized using microbatch-under-oil employing the random microseed matrix screening method. The protein crystallized in space groupP21, with unit-cell parametersa= 77.6,b= 189.6,c= 120.4 Å, β = 108.4°. A Matthews coefficient of 2.89 Å3 Da−1corresponds to four monomers, each with a molecular mass of ∼73 kDa, in the asymmetric unit. The APHdr structure will reveal the mechanisms of substrate selectivity and active-site accessibility in the bacterial enzyme. It will also be helpful in elucidating the functional role of this enzyme inD. radiodurans.

scholarly journals Active site geometry of oxalate decarboxylase from Flammulina velutipes: Role of histidine-coordinated manganese in substrate recognition

2009 ◽  
Vol 11 (9) ◽  
pp. 2138-2147 ◽  
Author(s):  
Subhra Chakraborty ◽  
Niranjan Chakraborty ◽  
Deepti Jain ◽  
Dinakar M. Salunke ◽  
Asis Datta
Keyword(s):  
Active Site ◽  
Substrate Recognition ◽  
Flammulina Velutipes ◽  
Oxalate Decarboxylase

scholarly journals Role of Arginine 117 in Substrate Recognition by Human Cytochrome P450 2J2

2018 ◽  
Vol 19 (7) ◽  
pp. 2066 ◽  
Author(s):  
Pierre Lafite ◽  
François André ◽  
Joan Graves ◽  
Darryl Zeldin ◽  
Patrick Dansette ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Active Site ◽  
Substrate Recognition ◽  
Salt Bridge ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Human Cytochrome ◽  
Dynamic Docking ◽  
Homology Models

The influence of Arginine 117 of human cytochrome P450 2J2 in the recognition of ebastine and a series of terfenadone derivatives was studied by site-directed mutagenesis. R117K, R117E, and R117L mutants were produced, and the behavior of these mutants in the hydroxylation of ebastine and terfenadone derivatives was compared to that of wild-type CYP2J2. The data clearly showed the importance of the formation of a hydrogen bond between R117 and the keto group of these substrates. The data were interpreted on the basis of 3D homology models of the mutants and of dynamic docking of the substrates in their active site. These modeling studies also suggested the existence of a R117-E222 salt bridge between helices B’ and F that would be important for maintaining the overall folding of CYP2J2.

scholarly journals Oligoribonuclease functions as a diribonucleotidase to bypass a key bottleneck in RNA degradation

2019 ◽  
Author(s):  
Soo-Kyoung Kim ◽  
Justin D. Lormand ◽  
Cordelia A. Weiss ◽  
Karin A. Eger ◽  
Husan Turdiev ◽  
...  
Keyword(s):  
Active Site ◽  
Rna Degradation ◽  
Degradation Pathway ◽  
Substrate Selectivity ◽  
Cellular Physiology ◽  
Functional Studies ◽  
Multistep Process ◽  
Rna Fragments ◽  
Mechanistic Basis ◽  
The One

AbstractDegradation of RNA polymers is a multistep process catalyzed by specific subsets of RNases. In all cases, degradation is completed by exoribonucleases that recycle RNA fragments into nucleotide monophosphate. In γ-proteobacteria, a group of up to eight 3’-5’ exoribonucleases have been implicated in RNA degradation. Oligoribonuclease (Orn) is unique among them as its activity is required for clearing short RNA fragments, a function important for cellular fitness. However, the mechanistic basis for this substrate selectivity remained unclear. Here we show that Orn’s activity as a general exoribonuclease has been vastly overestimated by demonstrating that the enzyme exhibits a much narrower substrate preference for diribonucleotides. Co-crystal structures of Orn with substrates reveal an active site optimized for diribonucleotides that does not accommodate longer substrates. While other cellular RNases process oligoribonucleotides down to diribonucleotide entities, our functional studies demonstrate that Orn is the one and only diribonucleotidase that completes the final stage of the RNA degradation pathway. Together, these results indicate that Orn is a dedicated diribonucleotidase that clears the diribonucleotide pool that otherwise affects cellular physiology and viability.

Important functional role of residue x of the presenilin GxGD protease active site motif for APP substrate cleavage specificity and substrate selectivity of γ-secretase

2013 ◽  
Vol 125 (1) ◽  
pp. 144-156 ◽  
Author(s):  
Benedikt Kretner ◽  
Akio Fukumori ◽  
Peer-Hendrik Kuhn ◽  
Blanca Isabel Pérez-Revuelta ◽  
Stefan F. Lichtenthaler ◽  
...  
Keyword(s):  
Active Site ◽  
Functional Role ◽  
Substrate Selectivity ◽  
Cleavage Specificity ◽  
Active Site Motif ◽  
Substrate Cleavage

Heparan sulphate N-sulphotransferase activity: reaction mechanism and substrate recognition

2003 ◽  
Vol 31 (2) ◽  
pp. 331-334 ◽  
Author(s):  
Y. Kakuta ◽  
L. Li ◽  
L.C. Pedersen ◽  
L.G. Pedersen ◽  
M. Negishi
Keyword(s):  
Active Site ◽  
Transfer Reaction ◽  
Substrate Recognition ◽  
Heparan Sulphate ◽  
Critical Role ◽  
Random Coil ◽  
Group Transfer ◽  
Α Helix ◽  
Model Chain

Human heparan sulphate N-deacetylase/N-sulphotransferase 1 sulphates the NH3+ group of the glucosamine moiety of the heparan chain in heparan sulphate/heparin biosynthesis. An open cleft that runs perpendicular to the sulphate donor 3´-phosphoadenosine 5´-phosphosulphate may constitute the acceptor substrate-binding site of the sulphotransferase domain (hNST1) [Kakuta, Sueyoshi, Negishi and Pedersen (1999) J. Biol. Chem. 274, 10673–10676]. When a hexasaccharide model chain is docked into the active site, only a trisaccharide (-IdoA-GlcN-IdoA-) portion interacts directly with the cleft residues: Trp-713, His-716 and His-720 from α helix 6, and Phe-640, Glu-641, Glu-642, Gln-644 and Asn-647 from random coil (residues 640–647). Mutation of these residues either abolishes or greatly reduces hNST1 activity. Glu-642 may play the critical role of catalytic base in the sulphuryl group transfer reaction, as indicated by its hydrogen-bonding distance to the NH3+ group of the glucosamine moiety in the model and by mutational data.

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