Complete sequence-specific1H NMR resonance assignment of hyperfine-shifted residues in the active site of a paramagnetic protein: Application toAplysia cyano-metmyoglobin

1992 ◽  
Vol 2 (6) ◽  
pp. 597-618 ◽  
Author(s):  
Jun Qin ◽  
Gerd N. La Mar
Keyword(s):  
Resonance Assignment ◽  
Active Site ◽  
Complete Sequence ◽  
Nmr Resonance Assignment ◽  
Paramagnetic Protein

scholarly journals Mechanistic and active-site studies on d(–)-mandelate dehydrogenase from Rhodotorula graminis

1992 ◽  
Vol 281 (1) ◽  
pp. 211-218 ◽  
Author(s):  
D P Baker ◽  
C Kleanthous ◽  
J N Keen ◽  
E Weinhold ◽  
C A Fewson
Keyword(s):  
Active Site ◽  
Complete Sequence ◽  
Histidine Residue ◽  
Extended Version ◽  
Order Process ◽  
First Order ◽  
Tryptic Digest ◽  
Complete Inactivation ◽  
Hydrolysis Of ◽  
High Voltage Electrophoresis

D(–)-Mandelate dehydrogenase, the first enzyme of the mandelate pathway in the yeast Rhodotorula graminis, catalyses the NAD(+)-dependent oxidation of D(–)-mandelate to phenylglyoxylate. D(–)-2-(Bromoethanoyloxy)-2-phenylethanoic acid [‘D(–)-bromoacetylmandelic acid’], an analogue of the natural substrate, was synthesized as a probe for reactive and accessible nucleophilic groups within the active site of the enzyme. D(–)-Mandelate dehydrogenase was inactivated by D(–)-bromoacetylmandelate in a psuedo-first-order process. D(–)-Mandelate protected against inactivation, suggesting that the residue that reacts with the inhibitor is located at or near the active site. Complete inactivation of the enzyme resulted in the incorporation of approx. 1 mol of label/mol of enzyme subunit. D(–)-Mandelate dehydrogenase that had been inactivated with 14C-labelled D(–)-bromoacetylmandelate was digested with trypsin; there was substantial incorporation of 14C into two tryptic-digest peptides, and this was lowered in the presence of substrate. One of the tryptic peptides had the sequence Val-Xaa-Leu-Glu-Ile-Gly-Lys, with the residue at the second position being the site of radiolabel incorporation. The complete sequence of the second peptide was not determined, but it was probably an N-terminally extended version of the first peptide. High-voltage electrophoresis of the products of hydrolysis of modified protein showed that the major peak of radioactivity co-migrated with N tau-carboxymethylhistidine, indicating that a histidine residue at the active site of the enzyme is the most likely nucleophile with which D(–)-bromoacetylmandelate reacts. D(–)-Mandelate dehydrogenase was incubated with phenylglyoxylate and either (4S)-[4-3H]NADH or (4R)-[4-3H]NADH and then the resulting D(–)-mandelate and NAD+ were isolated. The enzyme transferred the pro-R-hydrogen atom from NADH during the reduction of phenylglyoxylate. The results are discussed with particular reference to the possibility that this enzyme evolved by the recruitment of a 2-hydroxy acid dehydrogenase from another metabolic pathway.

scholarly journals Erratum to: NMR resonance assignment of DnaE intein from Nostoc punctiforme

2012 ◽  
Vol 7 (1) ◽  
pp. 115-116
Author(s):  
Kimmo Heinämäki ◽  
Jesper S. Oeemig ◽  
Kimmo Pääkkönen ◽  
Janica Djupsjöbacka ◽  
Hideo Iwaï
Keyword(s):  
Resonance Assignment ◽  
Nmr Resonance Assignment ◽  
Nostoc Punctiforme

scholarly journals NMR resonance assignment and dynamics of profilin from Heimdallarchaeota

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Syed Razaul Haq ◽  
Sabeen Survery ◽  
Fredrik Hurtig ◽  
Ann-Christin Lindås ◽  
Celestine N. Chi
Keyword(s):  
Structural Analysis ◽  
Structural Properties ◽  
Resonance Assignment ◽  
Eukaryotic Cell ◽  
Nmr Resonance Assignment ◽  
Metagenomic Data ◽  
Divergent Evolution ◽  
Structural Elements ◽  
Scientific Question ◽  
Secondary Structural Elements

Abstract The origin of the eukaryotic cell is an unsettled scientific question. The Asgard superphylum has emerged as a compelling target for studying eukaryogenesis due to the previously unseen diversity of eukaryotic signature proteins. However, our knowledge about these proteins is still relegated to metagenomic data and very little is known about their structural properties. Additionally, it is still unclear if these proteins are functionally homologous to their eukaryotic counterparts. Here, we expressed, purified and structurally characterized profilin from Heimdallarchaeota in the Asgard superphylum. The structural analysis shows that while this profilin possesses similar secondary structural elements as eukaryotic profilin, it contains additional secondary structural elements that could be critical for its function and an indication of divergent evolution.

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