scholarly journals Structural basis of light-induced redox regulation in the Calvin cycle

2018 ◽  
Author(s):  
Ciaran McFarlane ◽  
Nita R. Shah ◽  
Burak V. Kabasakal ◽  
Charles A.R. Cotton ◽  
Doryen Bubeck ◽  
...  
Keyword(s):  
Carbon Dioxide ◽  
Active Site ◽  
Active Sites ◽  
Redox State ◽  
Redox Regulation ◽  
Carbon Fixation ◽  
Calvin Cycle ◽  
Structural Basis ◽  
Disordered Protein ◽  
Calvin Cycle Enzymes

AbstractIn plants, carbon dioxide is fixed via the Calvin cycle in a tightly regulated process. Key to this regulation is the conditionally disordered protein CP12. CP12 forms a complex with two Calvin cycle enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), inhibiting their activities. The mode of CP12 action was unknown. By solving crystal structures of CP12 bound to GAPDH, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide pre-orders CP12 prior to binding the PRK active site. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our model explains how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.One Sentence SummaryHow plants turn off carbon fixation in the dark.

scholarly journals Structural basis of light-induced redox regulation in the Calvin–Benson cycle in cyanobacteria

2019 ◽  
Vol 116 (42) ◽  
pp. 20984-20990 ◽  
Author(s):  
Ciaran R. McFarlane ◽  
Nita R. Shah ◽  
Burak V. Kabasakal ◽  
Blanca Echeverria ◽  
Charles A. R. Cotton ◽  
...  
Keyword(s):  
Active Sites ◽  
Redox State ◽  
Redox Regulation ◽  
Carbon Fixation ◽  
Structural Basis ◽  
Exit Point ◽  
Reduction Step ◽  
Ribulose Bisphosphate ◽  
Disordered Protein ◽  
Cycle Regulation

Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin–Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are essential CB-cycle enzymes that control substrate availability for the carboxylation enzyme Rubisco. PRK consumes ATP to produce the Rubisco substrate ribulose bisphosphate (RuBP). GAPDH catalyzes the reduction step of the CB cycle with NADPH to produce the sugar glyceraldehyde 3-phosphate (GAP), which is used for regeneration of RuBP and is the main exit point of the cycle. GAPDH and PRK are coregulated by the redox state of a conditionally disordered protein CP12, which forms a ternary complex with both enzymes. However, the structural basis of CB-cycle regulation by CP12 is unknown. Here, we show how CP12 modulates the activity of both GAPDH and PRK. Using thermophilic cyanobacterial homologs, we solve crystal structures of GAPDH with different cofactors and CP12 bound, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide preorders CP12 prior to binding the PRK active site, which is resolved in complex with CP12. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.

scholarly journals The Uncommon Active Site of D-Amino Acid Transaminase from Haliscomenobacter hydrossis: Biochemical and Structural Insights into the New Enzyme

Molecules ◽  
2021 ◽  
Vol 26 (16) ◽  
pp. 5053
Author(s):  
Alina K. Bakunova ◽  
Alena Yu. Nikolaeva ◽  
Tatiana V. Rakitina ◽  
Tatiana Y. Isaikina ◽  
Maria G. Khrenova ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Structural Analysis ◽  
Active Site ◽  
Active Sites ◽  
Structural Basis ◽  
Active Site Residues ◽  
Type Iv ◽  
Fold Type ◽  
Arginine Residues

Among industrially important pyridoxal-5’-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes.

scholarly journals High-Resolution Crystal Structure of the Subclass B3 Metallo-β-Lactamase BJP-1: Rational Basis for Substrate Specificity and Interaction with Sulfonamides

2010 ◽  
Vol 54 (10) ◽  
pp. 4343-4351 ◽  
Author(s):  
Jean-Denis Docquier ◽  
Manuela Benvenuti ◽  
Vito Calderone ◽  
Magdalena Stoczko ◽  
Nicola Menciassi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Bradyrhizobium Japonicum ◽  
Active Sites ◽  
Structural Diversity ◽  
Binding Pocket ◽  
Structural Basis ◽  
Side Chain ◽  
Functional Heterogeneity ◽  
Hydrophobic Binding

ABSTRACT Metallo-β-lactamases (MBLs) are important enzymatic factors in resistance to β-lactam antibiotics that show important structural and functional heterogeneity. BJP-1 is a subclass B3 MBL determinant produced by Bradyrhizobium japonicum that exhibits interesting properties. BJP-1, like CAU-1 of Caulobacter vibrioides, overall poorly recognizes β-lactam substrates and shows an unusual substrate profile compared to other MBLs. In order to understand the structural basis of these properties, the crystal structure of BJP-1 was obtained at 1.4-Å resolution. This revealed significant differences in the conformation and locations of the active-site loops, determining a rather narrow active site and the presence of a unique N-terminal helix bearing Phe-31, whose side chain binds in the active site and represents an obstacle for β-lactam substrate binding. In order to probe the potential of sulfonamides (known to inhibit various zinc-dependent enzymes) to bind in the active sites of MBLs, the structure of BJP-1 in complex with 4-nitrobenzenesulfonamide was also obtained (at 1.33-Å resolution), thereby revealing the mode of interaction of these molecules in MBLs. Interestingly, sulfonamide binding resulted in the displacement of the side chain of Phe-31 from its hydrophobic binding pocket, where the benzene ring of the molecule is now found. These data further highlight the structural diversity shown by MBLs but also provide interesting insights in the structure-function relationships of these enzymes. More importantly, we provided the first structural observation of MBL interaction with sulfonamides, which might represent an interesting scaffold for the design of MBL inhibitors.

The LarB carboxylase/hydrolase forms a transient cysteinyl-pyridine intermediate during nickel-pincer nucleotide cofactor biosynthesis

2021 ◽  
Vol 118 (39) ◽  
pp. e2106202118
Author(s):  
Joel A. Rankin ◽  
Shramana Chatterjee ◽  
Zia Tariq ◽  
Satyanarayana Lagishetty ◽  
Benoît Desguin ◽  
...  
Keyword(s):  
Carbon Dioxide ◽  
Active Site ◽  
Active Sites ◽  
Pyridine Ring ◽  
Structural Investigations ◽  
Terminal Domain ◽  
Covalent Adduct ◽  
Pyridinium Ring ◽  
Cofactor Biosynthesis ◽  
Enzymatic Mechanisms

Enzymes possessing the nickel-pincer nucleotide (NPN) cofactor catalyze C2 racemization or epimerization reactions of α-hydroxyacid substrates. LarB initiates synthesis of the NPN cofactor from nicotinic acid adenine dinucleotide (NaAD) by performing dual reactions: pyridinium ring C5 carboxylation and phosphoanhydride hydrolysis. Here, we show that LarB uses carbon dioxide, not bicarbonate, as the substrate for carboxylation and activates water for hydrolytic attack on the AMP-associated phosphate of C5-carboxylated-NaAD. Structural investigations show that LarB has an N-terminal domain of unique fold and a C-terminal domain homologous to aminoimidazole ribonucleotide carboxylase/mutase (PurE). Like PurE, LarB is octameric with four active sites located at subunit interfaces. The complex of LarB with NAD+, an analog of NaAD, reveals the formation of a covalent adduct between the active site Cys221 and C4 of NAD+, resulting in a boat-shaped dearomatized pyridine ring. The formation of such an intermediate with NaAD would enhance the reactivity of C5 to facilitate carboxylation. Glu180 is well positioned to abstract the C5 proton, restoring aromaticity as Cys221 is expelled. The structure of as-isolated LarB and its complexes with NAD+ and the product AMP identify additional residues potentially important for substrate binding and catalysis. In combination with these findings, the results from structure-guided mutagenesis studies lead us to propose enzymatic mechanisms for both the carboxylation and hydrolysis reactions of LarB that are distinct from that of PurE.

scholarly journals Structural Basis for the Selective Inhibition of Cdc2-Like Kinases by CX-4945

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Joo Youn Lee ◽  
Ji-Sook Yun ◽  
Woo-Keun Kim ◽  
Hang-Suk Chun ◽  
Hyeonseok Jin ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Hydrophobic Interactions ◽  
Genetic Diseases ◽  
Selective Inhibition ◽  
Inhibitory Effect ◽  
Structural Basis ◽  
Active Site Pocket ◽  
Strong Inhibitory Effect ◽  
Interacting Networks

Cdc2-like kinases (CLKs) play a crucial role in the alternative splicing of eukaryotic pre-mRNAs through the phosphorylation of serine/arginine-rich proteins (SR proteins). Dysregulation of this processes is linked with various diseases including cancers, neurodegenerative diseases, and many genetic diseases. Thus, CLKs have been regarded to have a potential as a therapeutic target and significant efforts have been exerted to discover an effective inhibitor. In particular, the small molecule CX-4945, originally identified as an inhibitor of casein kinase 2 (CK2), was further revealed to have a strong CLK-inhibitory activity. Four isoforms of CLKs (CLK1, CLK2, CLK3, and CLK4) can be inhibited by CX-4945, with the highest inhibitory effect on CLK2. This study aimed to elucidate the structural basis of the selective inhibitory effect of CX-4945 on different isoforms of CLKs. We determined the crystal structures of CLK1, CLK2, and CLK3 in complex with CX-4945 at resolutions of 2.4 Å, 2.8 Å, and 2.6 Å, respectively. Comparative analysis revealed that CX-4945 was bound in the same active site pocket of the CLKs with similar interacting networks. Intriguingly, the active sites of CLK/CX-4945 complex structures had different sizes and electrostatic surface charge distributions. The active site of CLK1 was somewhat narrow and contained a negatively charged patch. CLK3 had a protruded Lys248 residue in the entrance of the active site pocket. In addition, Ala319, equivalent to Val324 (CLK1) and Val326 (CLK2), contributed to the weak hydrophobic interactions with the benzonaphthyridine ring of CX-4945. In contrast, the charge distribution pattern of CLK2 was the weakest, favoring its interactions with benzonaphthyridine ring. Thus, the relatively strong binding affinities of CX-4945 with CLK2 are consistent with its strong inhibitory effect defined in the previous study. These results may provide insights into structure-based drug discovery processes.

scholarly journals Carbon dioxide fixation by Calvin-Cycle enzymes improves ethanol yield in yeast

2013 ◽  
Vol 6 (1) ◽  
pp. 125 ◽  
Author(s):  
Víctor Guadalupe-Medina ◽  
H Wisselink ◽  
Marijke AH Luttik ◽  
Erik de Hulster ◽  
Jean-Marc Daran ◽  
...  
Keyword(s):  
Carbon Dioxide ◽  
Ethanol Yield ◽  
Calvin Cycle ◽  
Carbon Dioxide Fixation ◽  
Calvin Cycle Enzymes

scholarly journals Structural adaptation of oxygen tolerance in 4-hydroxybutyrl-CoA dehydratase, a key enzyme of archaeal carbon fixation

2020 ◽  
Author(s):  
Hasan DeMirci ◽  
Bradley B. Tolar ◽  
Tzanko Doukov ◽  
Aldis Petriceks ◽  
Akshaye Pal ◽  
...  
Keyword(s):  
Active Site ◽  
Carbon Fixation ◽  
Enzyme Stability ◽  
Structural Basis ◽  
Life Strategy ◽  
Structural Adaptation ◽  
Oxygen Tolerance ◽  
Ammonia Oxidizing Archaea ◽  
Oxygen Sensitive ◽  
Key Enzyme

AbstractAutotrophic microorganisms that convert inorganic carbon into organic matter were key players in the evolution of life on Earth. As the early atmosphere became oxygenated, microorganisms needed to develop mechanisms for oxygen protection, especially those relying on enzymes containing oxygen-sensitive metal clusters (e.g., Fe-S). Here we investigated how 4-hydroxybutyryl-CoA dehydratase (4HBD) - the key enzyme of the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle for CO2-fixation - adapted as conditions shifted from anoxic to oxic. 4HBD is found in both anaerobic bacteria and aerobic ammonia-oxidizing archaea (AOA). The oxygen-sensitive bacterial 4HBD and oxygen-tolerant archaeal 4HBD share 59 % amino acid identity. To examine the structural basis of oxygen tolerance in archaeal 4HBD, we determined the atomic resolution structure of the enzyme. Two tunnels providing access to the canonical [4Fe-4S] cluster in oxygen-sensitive bacterial 4HBD were closed with four conserved mutations found in all aerobic AOA and other archaea. Further biochemical experiments and molecular dynamics simulations support our findings that restricting access to the active site is the key to oxygen tolerance, explaining how active site evolution drove a major evolutionary transition.Significance statementAutotrophy (primary production) was the first life strategy on Earth. Before photosynthesis (using solar energy to fix carbon dioxide), life relied on chemical reactions for energy. These chemosynthetic reactions are present in all domains of life, including archaea possessing the most energy-efficient carbon fixation pathway - the 3-hydroxypropionate/4-hydroxybutyrate cycle. This efficiency results from enzyme modifications, including enhanced enzyme stability and catalysis of multiple reactions. We reveal the first structure of aerobic 4-hydroxybutyryl-CoA dehydratase (4HBD) from ammonia-oxidizing archaea. These archaea are among the most abundant organisms on the planet, and their 4HBD active site evolved oxygen tolerance to support aerobic metabolism. This modification can provide further insight into enzyme evolution on early earth, as photosynthesis developed and began oxygenating the atmosphere.

scholarly journals Structural basis for the hydrolytic dehalogenation of the fungicide chlorothalonil

2020 ◽  
Vol 295 (26) ◽  
pp. 8668-8677
Author(s):  
Daniel S. Catlin ◽  
Xinhang Yang ◽  
Brian Bennett ◽  
Richard C. Holz ◽  
Dali Liu
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Bound Water ◽  
Structural Basis ◽  
X Ray ◽  
Distorted Trigonal Bipyramid ◽  
Industrial Compounds ◽  
Structural Site ◽  
Trigonal Bipyramid Geometry

Cleavage of aromatic carbon–chlorine bonds is critical for the degradation of toxic industrial compounds. Here, we solved the X-ray crystal structure of chlorothalonil dehalogenase (Chd) from Pseudomonas sp. CTN-3, with 15 of its N-terminal residues truncated (ChdT), using single-wavelength anomalous dispersion refined to 1.96 Å resolution. Chd has low sequence identity (<15%) compared with all other proteins whose structures are currently available, and to the best of our knowledge, we present the first structure of a Zn(II)-dependent aromatic dehalogenase that does not require a coenzyme. ChdT forms a “head-to-tail” homodimer, formed between two α-helices from each monomer, with three Zn(II)-binding sites, two of which occupy the active sites, whereas the third anchors a structural site at the homodimer interface. The catalytic Zn(II) ions are solvent-accessible via a large hydrophobic (8.5 × 17.8 Å) opening to bulk solvent and two hydrophilic branched channels. Each active-site Zn(II) ion resides in a distorted trigonal bipyramid geometry with His117, His257, Asp116, Asn216, and a water/hydroxide as ligands. A conserved His residue, His114, is hydrogen-bonded to the Zn(II)-bound water/hydroxide and likely functions as the general acid-base. We examined substrate binding by docking chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile, TPN) into the hydrophobic channel and observed that the most energetically favorable pose includes a TPN orientation that coordinates to the active-site Zn(II) ions via a CN and that maximizes a π–π interaction with Trp227. On the basis of these results, along with previously reported kinetics data, we propose a refined catalytic mechanism for Chd-mediated TPN dehalogenation.

scholarly journals Structural basis for the function and regulation of the receptor protein tyrosine phosphatase CD45

2005 ◽  
Vol 201 (3) ◽  
pp. 441-452 ◽  
Author(s):  
Hyun-Joo Nam ◽  
Florence Poy ◽  
Haruo Saito ◽  
Christin A. Frederick
Keyword(s):  
Crystal Structure ◽  
Protein Tyrosine Phosphatase ◽  
Active Site ◽  
Active Sites ◽  
Hydrophobic Interactions ◽  
Tyrosine Phosphatase ◽  
Receptor Protein ◽  
Structural Basis ◽  
Salt Bridges ◽  
Protein Tyrosine

CD45 is the prototypic member of transmembrane receptor-like protein tyrosine phosphatases (RPTPs) and has essential roles in immune functions. The cytoplasmic region of CD45, like many other RPTPs, contains two homologous protein tyrosine phosphatase domains, active domain 1 (D1) and catalytically impaired domain 2 (D2). Here, we report crystal structure of the cytoplasmic D1D2 segment of human CD45 in native and phosphotyrosyl peptide-bound forms. The tertiary structures of D1 and D2 are very similar, but doubly phosphorylated CD3ζ immunoreceptor tyrosine-based activation motif peptide binds only the D1 active site. The D2 “active site” deviates from the other active sites significantly to the extent that excludes any possibility of catalytic activity. The relative orientation of D1 and D2 is very similar to that observed in leukocyte common antigen–related protein with both active sites in an open conformation and is restrained through an extensive network of hydrophobic interactions, hydrogen bonds, and salt bridges. This crystal structure is incompatible with the wedge model previously suggested for CD45 regulation.

scholarly journals Unravelling the regulation pathway of photosynthetic AB-GAPDH

2021 ◽  
Author(s):  
Roberto Marotta ◽  
Alessandra Del Giudice ◽  
Gurrieri Libero ◽  
Silvia Fanti ◽  
Paolo Swec ◽  
...  
Keyword(s):  
Active Sites ◽  
Carbon Fixation ◽  
Environmental Changes ◽  
Enzyme Inactivation ◽  
Structural Basis ◽  
Reversible Inactivation ◽  
Cofactor Binding ◽  
B Subunit ◽  
Key Enzyme ◽  
B Subunits

Oxygenic phototrophs perform carbon fixation through the Calvin–Benson cycle. Different mechanisms adjust the cycle and the light–harvesting reactions to rapid environmental changes. Photosynthetic glyceraldehyde–3–phosphate dehydrogenase (GAPDH) is a key enzyme of the cycle. In land plants, different photosynthetic GAPDHs exist: the most abundant formed by hetero-tetramers of A and B–subunits, and the homotetramer A4. Regardless of the subunit composition, GAPDH is the major consumer of photosynthetic NADPH and for this reason is strictly regulated. While A4–GAPDH is regulated by CP12, AB–GAPDH is autonomously regulated through the C-terminal extension (CTE) of B–subunits. Reversible inactivation of AB–GAPDH occurs via oxidation of a cysteine pair located in the CTE, and substitution of NADP(H) with NAD(H) in the cofactor binding domain. These combined conditions lead to a change in the oligomerization state and enzyme inactivation. SEC–SAXS and single–particle cryoEM analysis disclosed the structural basis of this regulatory mechanism. Both approaches revealed that (A2B2)n–GAPDH oligomers with n=1, 2, 4 and 5 co–exist in a dynamic system. B–subunits mediate the contacts between adjacent A2B2 tetramers in A4B4 and A8B8 oligomers. The CTE of each B–subunit penetrates into the active site of a B–subunit of the adjacent tetramer, while the CTE of this subunit moves in the opposite direction, effectively preventing the binding of the substrate 1,3–bisphosphoglycerate in the B–subunits. The whole mechanism is made possible, and eventually controlled, by pyridine nucleotides. In fact, NAD(H) by removing NADP(H) from A–subunits allows the entrance of the CTE in B–subunits active sites and hence inactive oligomer stabilization.

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