scholarly journals Structural basis of glycerophosphodiester recognition by the Mycobacterium tuberculosis substrate-binding protein UgpB

2019 ◽  
Author(s):  
Jonathan Fenn ◽  
Ridvan Nepravishta ◽  
Collette S Guy ◽  
James Harrison ◽  
Jesus Angulo ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Potential Role ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
X Ray Crystallography ◽  
Biochemical Analyses ◽  
Substrate Binding Domain ◽  
Substrate Binding Protein

AbstractMycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and has evolved an incredible ability to survive latently within the human host for decades. The Mtb pathogen encodes for a low number of ATP-binding cassette (ABC) importers for the acquisition of carbohydrates that may reflect the nutrient poor environment within the host macrophages. Mtb UgpB (Rv2833) is the substrate binding domain of the UgpABCE transporter that recognises glycerophosphocholine (GPC) indicating a potential role in glycerophospholipid recycling. By using a combination of saturation transfer difference (STD) NMR and X-ray crystallography we report the structural analysis of Mtb UgpB complexed with GPC and have identified that Mtb UgpB is promiscuous for other glycerophosphodiesters. Complementary biochemical analyses and site-directed mutagenesis define the molecular basis and specificity of glycerophosphodiester recognition. Our results provide critical insights into the structural and functional role of the Mtb UgpB transporter and reveal that the specificity of this ABC-transporter is not limited to GPC therefore optimising the ability of Mtb to scavenge scarce nutrients and essential glycerophospholipid metabolites during intracellular infection.

scholarly journals Structural Basis of Glycerophosphodiester Recognition by the Mycobacterium tuberculosis Substrate-Binding Protein UgpB

2019 ◽  
Vol 14 (9) ◽  
pp. 1879-1887 ◽  
Author(s):  
Jonathan S. Fenn ◽  
Ridvan Nepravishta ◽  
Collette S. Guy ◽  
James Harrison ◽  
Jesus Angulo ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Binding Protein ◽  
Substrate Binding ◽  
Structural Basis ◽  
Substrate Binding Protein

scholarly journals A hybrid approach reveals the allosteric regulation of GTP cyclohydrolase I

2020 ◽  
Vol 117 (50) ◽  
pp. 31838-31849
Author(s):  
Rebecca Ebenhoch ◽  
Simone Prinz ◽  
Susann Kaltwasser ◽  
Deryck J. Mills ◽  
Robert Meinecke ◽  
...  
Keyword(s):  
Crystal Packing ◽  
Substrate Binding ◽  
Regulatory Protein ◽  
Allosteric Regulation ◽  
Hybrid Approach ◽  
Site Directed Mutagenesis ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
X Ray ◽  
X Ray Crystallography

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1−GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.

Structures of the substrate-binding protein provide insights into the multiple compatible solute binding specificities of the Bacillus subtilis ABC transporter OpuC

2011 ◽  
Vol 436 (2) ◽  
pp. 283-289 ◽  
Author(s):  
Yang Du ◽  
Wei-Wei Shi ◽  
Yong-Xing He ◽  
Yi-Hu Yang ◽  
Cong-Zhao Zhou ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Abc Transporter ◽  
Binding Protein ◽  
Substrate Binding ◽  
Compatible Solutes ◽  
Binding Pocket ◽  
Compatible Solute ◽  
Structural Basis ◽  
Binding Property ◽  
Substrate Binding Protein

The compatible solute ABC (ATP-binding cassette) transporters are indispensable for acquiring a variety of compatible solutes under osmotic stress in Bacillus subtilis. The substrate-binding protein OpuCC (Opu is osmoprotectant uptake) of the ABC transporter OpuC can recognize a broad spectrum of compatible solutes, compared with its 70% sequence-identical paralogue OpuBC that can solely bind choline. To explore the structural basis of this difference of substrate specificity, we determined crystal structures of OpuCC in the apo-form and in complex with carnitine, glycine betaine, choline and ectoine respectively. OpuCC is composed of two α/β/α globular sandwich domains linked by two hinge regions, with a substrate-binding pocket located at the interdomain cleft. Upon substrate binding, the two domains shift towards each other to trap the substrate. Comparative structural analysis revealed a plastic pocket that fits various compatible solutes, which attributes the multiple-substrate binding property to OpuCC. This plasticity is a gain-of-function via a single-residue mutation of Thr94 in OpuCC compared with Asp96 in OpuBC.

scholarly journals Molecular determinant of substrate binding and specificity of cytochrome P450 2J2

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Liang Xu ◽  
Liao Y. Chen
Keyword(s):  
Arachidonic Acid ◽  
Cytochrome P450 ◽  
Active Site ◽  
Structural Model ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Structural Basis ◽  
Molecular Determinant ◽  
Dynamics Simulations

AbstractCytochrome P450 2J2 (CYP2J2) is responsible for the epoxidation of endogenous arachidonic acid, and is involved in the metabolism of exogenous drugs. To date, no crystal structure of CYP2J2 is available, and the proposed structural basis for the substrate recognition and specificity in CYP2J2 varies with the structural models developed using different computational protocols. In this study, we developed a new structural model of CYP2J2, and explored its sensitivity to substrate binding by molecular dynamics simulations of the interactions with chemically similar fluorescent probes. Our results showed that the induced-fit binding of these probes led to the preferred active poses ready for the catalysis by CYP2J2. Divergent conformational dynamics of CYP2J2 due to the binding of each probe were observed. However, a stable hydrophobic clamp composed of residues I127, F310, A311, V380, and I487 was identified to restrict any substrate access to the active site of CYP2J2. Molecular docking of a series of compounds including amiodarone, astemizole, danazol, ebastine, ketoconazole, terfenadine, terfenadone, and arachidonic acid to CYP2J2 confirmed the role of those residues in determining substrate binding and specificity of CYP2J2. In addition to the flexibility of CYP2J2, the present work also identified other factors such as electrostatic potential in the vicinity of the active site, and substrate strain energy and property that have implications for the interpretation of CYP2J2 metabolism.

scholarly journals Cloning, Expression, and Site-Directed Mutagenesis of the Propene Monooxygenase Genes from Mycobacterium sp. Strain M156

2005 ◽  
Vol 71 (4) ◽  
pp. 1909-1914 ◽  
Author(s):  
Chan K. Chan Kwo Chion ◽  
Sarah E. Askew ◽  
David J. Leak
Keyword(s):  
Mutational Analysis ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
Styrene Oxidation ◽  
Overlapping Reading Frames ◽  
Reading Frames

ABSTRACT Propene monooxygenase has been cloned from Mycobacterium sp. strain M156, based on hybridization with the amoABCD genes of Rhodococcus corallinus B276. Sequencing indicated that the mycobacterial enzyme is a member of the binuclear nonheme iron monooxygenase family and, in gene order and sequence, is most similar to that from R. corallinus B-276. Attempts were made to express the pmoABCD operon in Escherichia coli and Mycobacterium smegmatis mc2155. In the former, there appeared to be a problem resolving overlapping reading frames between pmoA and -B and between pmoC and -D, while in the latter, problems were encountered with plasmid instability when the pmoABCD genes were placed under the control of the hsp60 heat shock promoter in the pNBV1 vector. Fortuitously, constructs with the opposite orientation were constitutively expressed at a level sufficient to allow preliminary mutational analysis. Two PMO active-site residues (A94 and V188) were targeted by site-directed mutagenesis to alter their stereoselectivity. The results suggest that changing the volume occupied by the side chain at V188 leads to a systematic alteration in the stereoselectivity of styrene oxidation, presumably by producing different orientations for substrate binding during catalysis. Changing the volume occupied by the side chain at A94 produced a nonsystematic change in stereoselectivity, which may be attributable to the role of this residue in expansion of the binding site during substrate binding. Neither set of mutations changed the enzyme's specificity for epoxidation.

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