scholarly journals Studies of the active site of m-calpain and the interaction with calpastatin

1993 ◽  
Vol 296 (1) ◽  
pp. 135-142 ◽  
Author(s):  
C Crawford ◽  
N R Brown ◽  
A C Willis
Keyword(s):  
Ion Exchange ◽  
Proteolytic Activity ◽  
Active Site ◽  
Binding Assay ◽  
Large Subunit ◽  
Gel Filtration ◽  
Small Subunit ◽  
Binding Domain ◽  
Competitive Binding Assay ◽  
Binding Domains

Calpain autolyses in the presence of Ca2+. In the case of m-calpain (80 + 30 kDa) the first product is an 80 + 18 kDa species which has an intact large subunit and the C-terminal Ca(2+)-binding domain of the small subunit. It was possible to bind E64 into the active site of calpain in the presence of Ca2+ before cleavage of either calpain subunit. This suggests that the active site is functional before any autolysis has occurred and that calpain is not a proenzyme. Prolonged autolysis generates several fragments including a 42 kDa active-site domain fragment that showed no proteolytic activity and Ca(2+)-binding domain fragments. Some of the Ca(2+)-binding domain fragments were found to exist as heterodimers (23 + 18 kDa and 22 + 18 kDa), with the Ca(2+)-binding domain of the large subunit interacting with the Ca(2+)-binding domain of the small subunit. These species were true heterodimers, as they showed co-elution of the two Ca(2+)-binding domains on ion-exchange and gel-filtration chromatography, and immunoprecipitation of both polypeptides with an antiserum specific for the small-subunit Ca(2+)-binding domain. The generation of the dimer species after only 15 min autolysis suggests that the interaction between the Ca(2+)-binding domains is present in the native calpain structure. The interaction of calpain with calpastatin was investigated using an assay based on binding to calpastatin-Sepharose and a competitive binding assay. Calpain, active-site-blocked calpain and calpain fragments generated by autolysis were studied. Calpain bound to calpastatin in the presence of Ca2+; however, the isolated active-site-containing 80 kDa large subunit (proteolytically inactive), a 42 kDa active-site-containing fragment (proteolytically inactive) and Ca(2+)-binding domain fragments of calpain did not. Active-site-blocked calpain bound to calpastatin, but with an affinity reduced by approximately two orders of magnitude when compared with native calpain.

scholarly journals A model system for [NiFe] hydrogenase maturation studies: Purification of an active site-containing hydrogenase large subunit without small subunit

FEBS Letters ◽  
2005 ◽  
Vol 579 (20) ◽  
pp. 4292-4296 ◽  
Author(s):  
Gordon Winter ◽  
Thorsten Buhrke ◽  
Oliver Lenz ◽  
Anne Katherine Jones ◽  
Michael Forgber ◽  
...  
Keyword(s):  
Active Site ◽  
Large Subunit ◽  
Small Subunit ◽  
Model System ◽  
Hydrogenase Maturation

scholarly journals Isolation, characterization and distribution of adenosine 3′:5′-cyclic monophosphate from Pinus radiata

1978 ◽  
Vol 175 (3) ◽  
pp. 931-936 ◽  
Author(s):  
T Wilson ◽  
E Moustafa ◽  
A G C Renwick
Keyword(s):  
Statistical Analysis ◽  
Cyclic Amp ◽  
Pinus Radiata ◽  
Degrees Of Freedom ◽  
Binding Assay ◽  
Gel Filtration ◽  
Plant Tissues ◽  
Competitive Binding Assay ◽  
Cyclic Monophosphate ◽  
Solvent Systems

Cyclic AMP was extracted in 0.1 M-HCl from tissues of Pinus radiata and purified by gel filtration on Sephadex G-10, and chromatography on Dowex AG1 (X2) and polyethyleneimine-cellulose in two separate solvent systems. Presumptive cyclic AMP from 10kg batches of pine needles was characterized by countercurrent distribution in the presence of cyclic [8-3H]AMP. Statistical analysis of the curves for radioactivity and mass (determined by the Gilman competitive-binding assay) showed that the fit of the curves was highly significant for seven degrees of freedom. The distribution of cyclic AMP within P. radiata and various other plant tissues was determined by the Gilman procedure. The results suggest that there is no relationship between variations in cyclic AMP concentrations and the known function of the tissue in which it was measured.

scholarly journals The Splicing Factor U2AF Small Subunit Is Functionally Conserved between Fission Yeast and Humans

2004 ◽  
Vol 24 (10) ◽  
pp. 4229-4240 ◽  
Author(s):  
Christopher J. Webb ◽  
Jo Ann Wise
Keyword(s):  
Rna Binding ◽  
Mutational Analysis ◽  
Large Subunit ◽  
Time Frame ◽  
Small Subunit ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Binding Domains ◽  
Site Recognition

ABSTRACT The small subunit of U2AF, which functions in 3′ splice site recognition, is more highly conserved than its heterodimeric partner yet is less thoroughly investigated. Remarkably, we find that the small subunit of Schizosaccharomyces pombe U2AF (U2AFSM) can be replaced in vivo by its human counterpart, demonstrating that the conservation extends to function. Precursor mRNAs accumulate in S. pombe following U2AFSM depletion in a time frame consistent with a role in splicing. A comprehensive mutational analysis reveals that all three conserved domains are required for viability. Notably, however, a tryptophan in the pseudo-RNA recognition motif implicated in a key contact with the large subunit by crystallographic data is dispensable whereas amino acids implicated in RNA recognition are critical. Mutagenesis of the two zinc-binding domains demonstrates that they are neither equivalent nor redundant. Finally, two- and three-hybrid analyses indicate that mutations with effects on large-subunit interactions are rare whereas virtually all alleles tested diminished RNA binding by the heterodimer. In addition to demonstrating extraordinary conservation of U2AF small-subunit function, these results provide new insights into the roles of individual domains and residues.

scholarly journals Spectroscopic Insights into the Oxygen-tolerant Membrane-associated [NiFe] Hydrogenase of Ralstonia eutropha H16

2009 ◽  
Vol 284 (24) ◽  
pp. 16264-16276 ◽  
Author(s):  
Miguel Saggu ◽  
Ingo Zebger ◽  
Marcus Ludwig ◽  
Oliver Lenz ◽  
Bärbel Friedrich ◽  
...  
Keyword(s):  
Active Site ◽  
Ralstonia Eutropha ◽  
Large Subunit ◽  
Magnetic Coupling ◽  
Spectroscopic Characterization ◽  
Spectroscopic Properties ◽  
Small Subunit ◽  
Reduced Form ◽  
Strictly Anaerobic ◽  
Redox States

This study provides the first spectroscopic characterization of the membrane-bound oxygen-tolerant [NiFe] hydrogenase (MBH) from Ralstonia eutropha H16 in its natural environment, the cytoplasmic membrane. The H2-converting MBH is composed of a large subunit, harboring the [NiFe] active site, and a small subunit, capable in coordinating one [3Fe4S] and two [4Fe4S] clusters. The hydrogenase dimer is electronically connected to a membrane-integral cytochrome b. EPR and Fourier transform infrared spectroscopy revealed a strong similarity of the MBH active site with known [NiFe] centers from strictly anaerobic hydrogenases. Most redox states characteristic for anaerobic [NiFe] hydrogenases were identified except for one remarkable difference. The formation of the oxygen-inhibited Niu-A state was never observed. Furthermore, EPR data showed the presence of an additional paramagnetic center at high redox potential (+290 mV), which couples magnetically to the [3Fe4S] center and indicates a structural and/or redox modification at or near the proximal [4Fe4S] cluster. Additionally, significant differences regarding the magnetic coupling between the Nia-C state and [4Fe4S] clusters were observed in the reduced form of the MBH. The spectroscopic properties are discussed with regard to the unusual oxygen tolerance of this hydrogenase and in comparison with those of the solubilized, dimeric form of the MBH.

scholarly journals The Epstein-Barr Virus (EBV) Deubiquitinating Enzyme BPLF1 Reduces EBV Ribonucleotide Reductase Activity

2009 ◽  
Vol 83 (9) ◽  
pp. 4345-4353 ◽  
Author(s):  
Christopher B. Whitehurst ◽  
Shunbin Ning ◽  
Gretchen L. Bentz ◽  
Florent Dufour ◽  
Edward Gershburg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Ribonucleotide Reductase ◽  
Active Site ◽  
Epstein Barr Virus ◽  
Reductase Activity ◽  
Large Subunit ◽  
Small Subunit ◽  
Deubiquitinating Enzyme ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT A newly discovered virally encoded deubiquitinating enzyme (DUB) is strictly conserved across the Herpesviridae. Epstein-Barr virus (EBV) BPLF1 encodes a tegument protein (3,149 amino acids) that exhibits deubiquitinating (DUB) activity that is lost upon mutation of the active-site cysteine. However, targets for the herpesviral DUBs have remained elusive. To investigate a predicted interaction between EBV BPLF1 and EBV ribonucleotide reductase (RR), a functional clone of the first 246 N-terminal amino acids of BPLF1 (BPLF1 1-246) was constructed. Immunoprecipitation verified an interaction between the small subunit of the viral RR2 and BPLF1 proteins. In addition, the large subunit (RR1) of the RR appeared to be ubiquitinated both in vivo and in vitro; however, ubiquitinated forms of the small subunit, RR2, were not detected. Ubiquitination of RR1 requires the expression of both subunits of the RR complex. Furthermore, coexpression of RR1 and RR2 with BPLF1 1-246 abolishes ubiquitination of RR1. EBV RR1, RR2, and BPLF1 1-246 colocalized to the cytoplasm in HEK 293T cells. Finally, expression of enzymatically active BPLF1 1-246 decreased RR activity, whereas a nonfunctional active-site mutant (BPLF1 C61S) had no effect. These results indicate that the EBV deubiquitinating enzyme interacts with, deubiquitinates, and influences the activity of the EBV RR. This is the first verified protein target of the EBV deubiquitinating enzyme.

scholarly journals Crystal structures of a [NiFe] hydrogenase large subunit HyhL in an immature state in complex with a Ni chaperone HypA

2018 ◽  
Vol 115 (27) ◽  
pp. 7045-7050 ◽  
Author(s):  
Sunghark Kwon ◽  
Satoshi Watanabe ◽  
Yuichi Nishitani ◽  
Takumi Kawashima ◽  
Tamotsu Kanai ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Structure Analysis ◽  
Active Site ◽  
Cleavage Site ◽  
Large Subunit ◽  
Binding Domain ◽  
Terminal Region ◽  
Cluster Assembly ◽  
Unknown Mechanism ◽  
Mature Form

Ni-Fe clusters are inserted into the large subunit of [NiFe] hydrogenases by maturation proteins such as the Ni chaperone HypA via an unknown mechanism. We determined crystal structures of an immature large subunit HyhL complexed with HypA from Thermococcus kodakarensis. Structure analysis revealed that the N-terminal region of HyhL extends outwards and interacts with the Ni-binding domain of HypA. Intriguingly, the C-terminal extension of immature HyhL, which is cleaved in the mature form, adopts a β-strand adjacent to its N-terminal β-strands. The position of the C-terminal extension corresponds to that of the N-terminal extension of a mature large subunit, preventing the access of endopeptidases to the cleavage site of HyhL. These findings suggest that Ni insertion into the active site induces spatial rearrangement of both the N- and C-terminal tails of HyhL, which function as a key checkpoint for the completion of the Ni-Fe cluster assembly.

Microbial hydrogen splitting in the presence of oxygen

2013 ◽  
Vol 41 (5) ◽  
pp. 1317-1324 ◽  
Author(s):  
Matthias Stein ◽  
Sandeep Kaur-Ghumaan
Keyword(s):  
Active Site ◽  
Large Subunit ◽  
Spectroscopic Studies ◽  
Small Subunit ◽  
Reduced Form ◽  
Cysteine Residues ◽  
Binding Motif ◽  
Oxygen Tolerance ◽  
Symmetry Approach ◽  
Long Time

The origin of the tolerance of a subclass of [NiFe]-hydrogenases to the presence of oxygen was unclear for a long time. Recent spectroscopic studies showed a conserved active site between oxygen-sensitive and oxygen-tolerant hydrogenases, and modifications in the vicinity of the active site in the large subunit could be excluded as the origin of catalytic activity even in the presence of molecular oxygen. A combination of bioinformatics and protein structural modelling revealed an unusual co-ordination motif in the vicinity of the proximal Fe–S cluster in the small subunit. Mutational experiments confirmed the relevance of two additional cysteine residues for the oxygen-tolerance. This new binding motif can be used to classify sequences from [NiFe]-hydrogenases according to their potential oxygen-tolerance. The X-ray structural analysis of the reduced form of the enzyme displayed a new type of [4Fe–3S] cluster co-ordinated by six surrounding cysteine residues in a distorted cubanoid geometry. The unusual electronic structure of the proximal Fe–S cluster can be analysed using the broken-symmetry approach and gave results in agreement with experimental Mößbauer studies. An electronic effect of the proximal Fe–S cluster on the remote active site can be detected and quantified. In the oxygen-tolerant hydrogenases, the hydride occupies an asymmetric binding position in the Ni-C state. This may rationalize the more facile activation and catalytic turnover in this subclass of enzymes.

scholarly journals Molybdenum-Containing Nicotine Hydroxylase Genes in a Nicotine Degradation Pathway That Is a Variant of the Pyridine and Pyrrolidine Pathways

2015 ◽  
Vol 81 (24) ◽  
pp. 8330-8338 ◽  
Author(s):  
Hao Yu ◽  
Hongzhi Tang ◽  
Yangyang Li ◽  
Ping Xu
Keyword(s):  
Inductively Coupled Plasma ◽  
Large Subunit ◽  
Sole Source ◽  
Small Subunit ◽  
Binding Domain ◽  
Mass Spectrometry Analysis ◽  
Resting Cells ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Nicotine Degradation

ABSTRACTOchrobactrumsp. strain SJY1 utilizes nicotine as a sole source of carbon, nitrogen, and energy via a variant of the pyridine and pyrrolidine pathways (the VPP pathway). Several strains and genes involved in the VPP pathway have recently been reported; however, the first catalyzing step for enzymatic turnover of nicotine is still unclear. In this study, a nicotine hydroxylase for the initial hydroxylation step of nicotine degradation was identified and characterized. The nicotine hydroxylase (VppA), which converts nicotine to 6-hydroxynicotine in the strain SJY1, is encoded by two open reading frames (vppASandvppAL[subunits S and L, respectively]). ThevppAgenes were heterologously expressed in the non-nicotine-degrading strainsEscherichia coliDH5α andPseudomonas putidaKT2440; only thePseudomonasstrain acquired the ability to degrade nicotine. The small subunit of VppA contained a [2Fe-2S] cluster-binding domain, and the large subunit of VppA contained a molybdenum cofactor-binding domain; however, an FAD-binding domain was not found in VppA. Resting cells cultivated in a molybdenum-deficient medium had low nicotine transformation activity, and excess molybdenum was detected in the purified VppA by inductively coupled plasma-mass spectrometry analysis. Thus, it is demonstrated that VppA is a two-component molybdenum-containing hydroxylase.

scholarly journals Investigation of the structural basis of the interaction of calpain II with phospholipid and with carbohydrate

1990 ◽  
Vol 265 (2) ◽  
pp. 575-579 ◽  
Author(s):  
C Crawford ◽  
N R Brown ◽  
A C Willis
Keyword(s):  
Large Subunit ◽  
Specific Interaction ◽  
Gel Filtration ◽  
Small Subunit ◽  
Complete Sequence ◽  
Structural Basis ◽  
Proteolytic Degradation ◽  
Amino Acid Sequence Analysis ◽  
Pig Kidney ◽  
Calpain Ii

Two forms of pig kidney calpain II were isolated, both of which appeared to contain an intact 80 kDa large subunit, but which showed specific proteolytic degradation at the N-terminal end of the 30 kDa small subunit. The structure of each of these molecules was investigated by amino acid sequence analysis. The forms corresponded to molecules with small subunits starting at residue 38 (degraded calpain A) and at residue 62 (degraded calpain B) of the complete sequence. These molecules were tested for their ability to interact with phosphatidylinositol and with carbohydrate (agarose gel-filtration media). Calpain and degraded calpain A, but not degraded calpain B, would interact with phosphatidylinositol. Thus the sequence (G)17TAMRILG (residues 38-61) is essential for the interaction. Neither calpain nor the degraded forms of the enzyme showed specific interaction with carbohydrate.

The importance of iron in the biosynthesis and assembly of [NiFe]-hydrogenases

2014 ◽  
Vol 5 (1) ◽  
pp. 55-70 ◽  
Author(s):  
Constanze Pinske ◽  
R. Gary Sawers
Keyword(s):  
Active Site ◽  
Large Subunit ◽  
Small Subunit ◽  
Transport Systems ◽  
Bacterial Cells ◽  
Carbamoyl Phosphate ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
Redox Active ◽  
Cofactor Biosynthesis

Abstract[NiFe]-hydrogenases (Hyd) are redox-active metalloenzymes that catalyze the reversible oxidation of molecular hydrogen to protons and electrons. These enzymes are frequently heterodimeric and have a unique bimetallic active site in their catalytic large subunit and possess a complement of iron sulfur (Fe-S) clusters for electron transfer in the small subunit. Depending on environmental and metabolic requirements, the Fe-S cluster relay shows considerable variation among the Hyd, even employing high potential [4Fe-3S] clusters for improved oxygen tolerance. The general iron sulfur cluster (Isc) machinery is required for small subunit maturation, possibly providing standard [4Fe-4S], which are then modified as required in situ. The [NiFe] cofactor in the active site also has an iron ion to which one CO and two CN- diatomic ligands are attached. Specific accessory proteins synthesize these ligands and insert the cofactor into the apo-hydrogenase large subunit. Carbamoyl phosphate is the precursor of the CN- ligands, and recent experimental evidence suggests that endogenously generated CO2 might be one precursor of CO. Recent advances also indicate how the machineries responsible for cofactor generation obtain iron. Several transport systems for iron into bacterial cells exist; however, in Escherichia coli, it is mainly the ferrous iron transporter Feo and the ferric-citrate siderphore system Fec that are involved in delivering the metal for Hyd biosynthesis. Genetic analyses have provided evidence for the existence of key checkpoints during cofactor biosynthesis and enzyme assembly that ensure correct spatiotemporal maturation of these modular oxidoreductases.

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