The structure, kinetics and interactions of the β-carboxysomal β-carbonic anhydrase, CcaA

2016 ◽  
Vol 473 (24) ◽  
pp. 4559-4572 ◽  
Author(s):  
Leah D. McGurn ◽  
Maryam Moazami-Goudarzi ◽  
Sean A. White ◽  
Tannu Suwal ◽  
Beant Brar ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Carbonic Anhydrase ◽  
Symmetry Axis ◽  
Proximal Part ◽  
Inhibit Activity ◽  
Binding Partners ◽  
Plant Enzymes ◽  
A Site ◽  
Catalytic Characterization ◽  
Synechocystis Sp

CcaA is a β-carbonic anhydrase (CA) that is a component of the carboxysomes of a subset of β-cyanobacteria. This protein, which has a characteristic C-terminal extension of unknown function, is recruited to the carboxysome via interactions with CcmM, which is itself a γ-CA homolog with enzymatic activity in many, but not all cyanobacteria. We have determined the structure of CcaA from Synechocystis sp. PCC 6803 at 1.45 Å. In contrast with the dimer-of-dimers organization of most bacterial β-CAs, or the loose dimer-of-dimers-of-dimers organization found in the plant enzymes, CcaA shows a well-packed trimer-of-dimers organization. The proximal part of the characteristic C-terminal extension is ordered by binding at a site that passes through the two-fold symmetry axis shared with an adjacent dimer; as a result, only one of a pair of converging termini can be ordered at any given time. Docking in Rosetta failed to find well-packed solutions, indicating that formation of the CcaA/CcmM complex probably requires significant backbone movements in at least one of the binding partners. Surface plasmon resonance experiments showed that CcaA forms a complex with CcmM with sub-picomolar affinity, with contributions from residues in CcmM's αA helix and CcaA's C-terminal tail. Catalytic characterization showed CcaA to be among the least active β-CAs characterized to date, with activity comparable with the γ-CA, CcmM, it either complements or replaces. Intriguingly, the C-terminal tail appears to partly inhibit activity, possibly indicating a role in minimizing the activity of unencapsulated enzyme.

Localization of Acidification of Urine, Potassium and Ammonia Secretion and Phosphate Reabsorption in the Nephron of the Dog

1958 ◽  
Vol 194 (1) ◽  
pp. 125-134 ◽  
Author(s):  
Robert F. Pitts ◽  
Ruth S. Gurd ◽  
Richard H. Kessler ◽  
Klaus Hierholzer
Keyword(s):  
Distal Part ◽  
Potassium Phosphate ◽  
Proximal Part ◽  
Ammonium Ion ◽  
Sodium Reabsorption ◽  
Hydrogen Ions ◽  
Acid Urine ◽  
Urine Potassium ◽  
A Site ◽  
Stop Flow

The Malvin, Sullivan and Wilde ( The Physiologist 1: 58, 1957) stop flow technique for the localization of tubular function has been applied to a study of potassium and acid excretion in the dog. It has been observed that the urine is acidified in the distal part of the nephron at a site of avid sodium reabsorption. Potassium and ammonia are secreted in the same portion of the tubule. Diamox reduces acidification of the urine and secretion of ammonia and enhances the secretion of potassium. Phosphate is reabsorbed in the proximal part of the nephron in a region which is coextensive with that which secretes p-aminohippurate. All our data are consonant with the view that a mechanism located in the distal part of the nephron exchanges cellular hydrogen and/or potassium ions for sodium ions in the tubular urine. Ammonia diffuses into acid urine and is trapped as ammonium ion. Diamox, by interfering with the supply of cellular hydrogen ions, reduces exchange of hydrogen for sodium and favors the exchange of potassium for sodium.

Acid secretion in isolated guinea pig colon

1987 ◽  
Vol 253 (2) ◽  
pp. G155-G164 ◽  
Author(s):  
Y. Suzuki ◽  
K. Kaneko
Keyword(s):  
Carbonic Anhydrase ◽  
Guinea Pig ◽  
Acid Secretion ◽  
Proximal Part ◽  
Bathing Solution ◽  
Maximal Effect ◽  
Dose Dependency ◽  
Ussing Chambers ◽  
Mucosal Acidification ◽  
Ph Stat

Isolated guinea pig distal colons secreted acid into the mucosal bathing solution at a rate of 1.0-1.5 mumol X cm-2 X h-1 when the preparations were mounted in Ussing chambers and bathed with HCO3(-)-CO2-free solution. The rates of the acidification and alkalinization of the solutions were measured by a pH stat system or calculated from changes in the pH of the solution. The acid secretion was localized in the middle and distal parts of the colon but absent in the proximal part of the colon and the cecum. The mucosal acidification was accompanied by serosal alkalinization, the rate of the latter being approximately 60% of the former. A carbonic anhydrase inhibitor, methazolamide (10(-4) M), reduced both the mucosal acidification and serosal alkalinization rates by a similar magnitude. The mucosal acidification was completely abolished by mucosal K+-free conditions but unaffected by mucosal Na+-free conditions. Ouabain added to the mucosal solution promptly inhibited the acid secretion. Dose dependency of the inhibition conformed to the Michaelis-Menten equation with a half-maximal effect at 4 X 10(-6) M. When the pH of the mucosal solution was reduced to 4.3, the rate of the mucosal acidification remained essentially the same as that at pH = 7.4. Vanadate (10(-4) M) added to both the mucosal and serosal solutions significantly reduced the mucosal acidification rate. These results suggest that CO2 derived from the epithelial metabolism is hydrated by carbonic anhydrase in the cell and released H+ enters the mucosal solution while HCO3- enters the serosal solution. H+ exit across the mucosal membrane may be mediated by H+-ATPase that is sensitive to ouabain.

Role of histidine 64 in the catalytic mechanism of human carbonic anhydrase II studied with a site-specific mutant

Biochemistry ◽  
1989 ◽  
Vol 28 (19) ◽  
pp. 7913-7918 ◽  
Author(s):  
Chingkuang Tu ◽  
David N. Silverman ◽  
Cecilia Forsman ◽  
Bengt Harald Jonsson ◽  
Sven Lindskog
Keyword(s):  
Carbonic Anhydrase ◽  
Catalytic Mechanism ◽  
Carbonic Anhydrase Ii ◽  
Site Specific ◽  
Human Carbonic Anhydrase ◽  
A Site

Characterization of the C-terminal extension of carboxysomal carbonic anhydrase from Synechocystis sp. PCC6803

2002 ◽  
Vol 29 (3) ◽  
pp. 183 ◽  
Author(s):  
Anthony K.-C. So ◽  
Swan S.-W. Cot ◽  
George S. Espie
Keyword(s):  
Carbonic Anhydrase ◽  
Protein Interaction ◽  
Recombinant Dna ◽  
Carbonic Anhydrases ◽  
Recombinant Dna Technology ◽  
Higher Plant ◽  
High Sequence Identity ◽  
Synechocystis Pcc6803 ◽  
Plant Enzymes ◽  
Genes Encoding

Sequence analysis of the carboxysomal carbonic anhydrase (CcaA) from Synechocystis PCC6803, Synechococcus PCC7942 and Nostoc ATCC29133, indicated high sequence identity to the β class of plant and bacterial carbonic anhydrases (CA), and conservation of the active site region. However, the cyanobacterial enzyme has a C-terminal extension of about 75 amino acids (aa) not found in the plant enzymes, and largely absent from other bacterial enzymes. Using recombinant DNA technology, genes encoding C-terminal truncation products of up to 127 aa were overexpressed in E. coli, and partially purified lysates were analysed for CA-mediated exchange of 18O between 13C18O2and H216O. Recombinant CcaA proteins with up to 60 aa removed (CcaAΔ60) were catalytically competent, but beyond this there was an abrupt loss of activity. CcaAΔ0, along with CcaAΔ40 and CcaAΔ60, also catalysed the hydrolysis of carbon oxysulfide (COS; an isoelectronic structural analogue of CO2), but CcaAΔ63 and CcaAΔ127 did not, indicating that truncations greater than 62 aa resulted in a general loss of catalytic competency. Analysis of protein-protein interaction using the yeast two-hybrid system revealed that CcaA did not interact with the large or small Rubisco subunits (RbcL and RbcS, respectively) of Synechocystis, but there was strong CcaA-CcaA interaction. This protein interaction also ceased with C-terminal truncations in CcaA greater than 60 aa. The correlation between loss of CcaA-CcaA interaction and CcaA catalytic activity suggests that the proximal portion of the C-terminal extension is required for oligomerization, and that this oligomerization is essential for catalysis by the cyanobacterial enzyme. Thus, the C-terminal extension may play an important role in the function of CA within cyanobacterial carboxysomes, which is not required by the higher plant enzymes.

Insight into calcification of Synechocystis sp. enhanced by extracellular carbonic anhydrase

RSC Advances ◽  
2016 ◽  
Vol 6 (35) ◽  
pp. 29811-29817 ◽  
Author(s):  
Zhen-Ni Yang ◽  
Xiao-Min Li ◽  
Ahmad Umar ◽  
Wen-Hong Fan ◽  
Yao Wang
Keyword(s):  
Carbon Dioxide ◽  
Carbonic Anhydrase ◽  
Insight Into ◽  
Synechocystis Sp

The mechanism of cyanobacterial calcification was proved to be related to extracellular carbonic anhydrase, which enhanced CaCO3 precipitation through facilitating proton consumption during transformation of bicarbonate to carbon dioxide.

scholarly journals Fusion Deficiency Induced by Mutations at the Dimer Interface in the Newcastle Disease Virus Hemagglutinin-Neuraminidase Is due to a Temperature-Dependent Defect in Receptor Binding

2003 ◽  
Vol 77 (12) ◽  
pp. 6913-6922 ◽  
Author(s):  
Elizabeth A. Corey ◽  
Anne M. Mirza ◽  
Elizabeth Levandowsky ◽  
Ronald M. Iorio
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Mutational Analysis ◽  
Proximal Part ◽  
Surface Glycoprotein ◽  
Crystallographic Structure ◽  
Dimer Interface ◽  
A Site ◽  
Viral Surface

ABSTRACT The tetrameric paramyxovirus hemagglutinin-neuraminidase (HN) protein mediates attachment to sialic acid-containing receptors as well as cleavage of the same moiety via its neuraminidase (NA) activity. The X-ray crystallographic structure of an HN dimer from Newcastle disease virus (NDV) suggests that a single site in two different conformations mediates both of these activities. This conformational change is predicted to involve an alteration in the association between monomers in each HN dimer and to be part of a series of changes in the structure of HN that link its recognition of receptors to the activation of the other viral surface glycoprotein, the fusion protein. To explore the importance of the dimer interface to HN function, we performed a site-directed mutational analysis of residues in a domain defined by residues 218 to 226 at the most membrane-proximal part of the dimer interface in the globular head. Proteins carrying substitutions for residues F220, S222, and L224 in this domain were fusion deficient. However, this fusion deficiency was not due to a direct effect of the mutations on fusion. Rather, the fusion defect was due to a severely impaired ability to mediate receptor recognition at 37°C, a phenotype that is not attributable to a change in NA activity. Since each of these mutated proteins efficiently mediated attachment in the cold, it was also not due to an inherent inability of the mutated proteins to recognize receptors. Instead, the interface mutations acted by weakening the interaction between HN and its receptor(s). The phenotype of these mutants correlates with the disruption of intermonomer subunit interactions.

scholarly journals SAM homeostasis is regulated by CFIm-mediated splicing of MAT2A

2020 ◽  
Author(s):  
Anna M. Scarborough ◽  
Juliana N. Flaherty ◽  
Olga V. Hunter ◽  
Kuanqing Liu ◽  
Ashwani Kumar ◽  
...  
Keyword(s):  
Site Selection ◽  
Binding Sites ◽  
Unknown Mechanism ◽  
Knock Out ◽  
Methyl Donor ◽  
Factor I ◽  
Binding Partners ◽  
Sam Synthetase ◽  
A Site ◽  
Rs Domains

SUMMARYS-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events. Cells regulate intracellular SAM levels through intron detention of the MAT2A RNA, which encodes only SAM synthetase expressed in most cells. The N6-adenosine methyltransferase METTL16 promotes splicing of the MAT2A detained intron by an unknown mechanism. Using an unbiased CRISPR knock-out screen, we identified CFIm25 (NUDT21) to be a regulator of MAT2A intron detention and intracellular SAM levels. CFIm25 is a component of the cleavage factor Im (CFIm) complex that regulates poly(A) site selection, but we show it promotes MAT2A splicing, independent of poly(A) site selection. CFIm25-mediated MAT2A splicing induction requires the RS domains of its binding partners, CFIm68 and CFIm59 as well as binding sites in detained intron and 3′ UTR. These studies uncover mechanisms that regulate MAT2A intron detention and reveal previously undescribed roles for CFIm in splicing and SAM metabolism.

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