Ribokinase family evolution and the role of conserved residues at the active site of the PfkB subfamily representative, Pfk-2 from Escherichia coli

ABSTRACT Amino acid substitutions in Escherichia coliς70 were generated and characterized in an analysis of the role of region 1.1 in transcription initiation. Several acidic and conserved residues are tolerant of substitution. However, replacement of aspartic acid 61 with alanine results in inactivity caused by structural and functional thermolability.

Download Full-text

GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation

Biochemical Journal ◽

10.1042/bj20090110 ◽

2009 ◽

Vol 420 (2) ◽

pp. 221-227 ◽

Cited By ~ 56

Author(s):

Helge C. Dorfmueller ◽

Vladimir S. Borodkin ◽

Marianne Schimpl ◽

Daan M. F. van Aalten

Keyword(s):

Active Site ◽

Cell Biology ◽

Rational Design ◽

Catalytic Mechanism ◽

Conserved Residues ◽

Cellular Processes ◽

Acetyl Group ◽

Nanomolar Range ◽

Insight Into

O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.

Download Full-text

Role of tryptophan 248 in the active site of tryptophanase from Escherichia coli

Biochemical and Biophysical Research Communications ◽

10.1016/s0006-291x(05)80100-7 ◽

1990 ◽

Vol 173 (2) ◽

pp. 756-762 ◽

Cited By ~ 4

Author(s):

Yasushi Kawata ◽

Nobuharu Tsujimoto ◽

Shunsuke Tani ◽

Tomohiro Mizobata ◽

Masanobu Tokushige

Keyword(s):

Escherichia Coli ◽

Active Site

Download Full-text

Functional role of a non-active site residue Trp23 on the enzyme activity of Escherichia coli thioesterase I/protease I/lysophospholipase L1

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics ◽

10.1016/j.bbapap.2009.06.008 ◽

2009 ◽

Vol 1794 (10) ◽

pp. 1467-1473 ◽

Cited By ~ 11

Author(s):

Li-Chiun Lee ◽

Yi-Li Chou ◽

Hong-Hwa Chen ◽

Ya-Lin Lee ◽

Jei-Fu Shaw

Keyword(s):

Escherichia Coli ◽

Enzyme Activity ◽

Active Site ◽

Functional Role ◽

Active Site Residue

Download Full-text

Analysis of the functional role of conserved residues in the protein subunit of ribonuclease P from Escherichia coli

Journal of Molecular Biology ◽

10.1006/jmbi.1997.0906 ◽

1997 ◽

Vol 267 (4) ◽

pp. 818-829 ◽

Cited By ~ 48

Author(s):

Venkat Gopalan ◽

Andreas D Baxevanis ◽

David Landsman ◽

Sidney Altman

Keyword(s):

Escherichia Coli ◽

Functional Role ◽

Ribonuclease P ◽

Protein Subunit ◽

Conserved Residues

Download Full-text

Serendipitous crystallization and structure determination of cyanase (CynS) fromSerratia proteamaculans

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15004902 ◽

2015 ◽

Vol 71 (4) ◽

pp. 471-476 ◽

Cited By ~ 4

Author(s):

Agata Butryn ◽

Gabriele Stoehr ◽

Christian Linke-Winnebeck ◽

Karl-Peter Hopfner

Keyword(s):

Escherichia Coli ◽

Carbon Dioxide ◽

Structure Determination ◽

Active Site ◽

Mass Spectrometric ◽

Mass Spectrometric Analysis ◽

Spectrometric Analysis ◽

Conserved Residues ◽

Serratia Proteamaculans

Cyanate hydratase (CynS) catalyzes the decomposition of cyanate and bicarbonate into ammonia and carbon dioxide. Here, the serendipitous crystallization of CynS fromSerratia proteamaculans(SpCynS) is reported. SpCynS was crystallized as an impurity and its identity was determined using mass-spectrometric analysis. The crystals belonged to space groupP1 and diffracted to 2.1 Å resolution. The overall structure of SpCynS is very similar to a previously determined structure of CynS fromEscherichia coli. Density for a ligand bound to the SpCynS active site was observed, but could not be unambiguously identified. Additionally, glycerol molecules bound at the entry to the active site of the enzyme indicate conserved residues that might be important for the trafficking of substrates and products.

Download Full-text

Structure and mechanism of the chalcogen-detoxifying protein TehB from Escherichia coli

Biochemical Journal ◽

10.1042/bj20102014 ◽

2011 ◽

Vol 435 (1) ◽

pp. 85-91 ◽

Cited By ~ 10

Author(s):

Hassanul G. Choudhury ◽

Alexander D. Cameron ◽

So Iwata ◽

Konstantinos Beis

Keyword(s):

Escherichia Coli ◽

Reaction Mechanism ◽

Active Site ◽

Kinetic Data ◽

Protein Analysis ◽

Nucleophilic Attack ◽

Conserved Residues ◽

Low Levels ◽

Living Organisms ◽

Derivatives Of

The oxyanion derivatives of the chalcogens tellurium and selenium are toxic to living organisms even at very low levels. Bacteria have developed mechanisms to overcome their toxicity by methylating them. The structure of TehB from Escherichia coli has been determined in the presence of the cofactor analogues SAH (S-adenosylhomocysteine) and sinefungin (a non-hydrolysable form of S-adenosyl-L-methionine) at 1.48 Å (1 Å=0.1 nm) and 1.9 Å respectively. Interestingly, our kinetic data show that TehB does not discriminate between selenium or tellurite oxyanions, making it a very powerful detoxifying protein. Analysis of the active site has identified three conserved residues that are capable of binding and orientating the metals for nucleophilic attack: His176, Arg177 and Arg184. Mutagenesis studies revealed that the H176A and R184A mutants retained most of their activity, whereas the R177A mutant had 65% of its activity abolished. Based on the structure and kinetic data we propose an SN2 nucleophilic attack reaction mechanism. These data provide the first molecular understanding of the detoxification of chalcogens by bacteria.

Download Full-text

Role of D-aminoacyl-tRNA deacylase beyond chiral proofreading as a cellular defense against glycine mischarging by AlaRS

eLife ◽

10.7554/elife.24001 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 19

Author(s):

Komal Ishwar Pawar ◽

Katta Suma ◽

Ayshwarya Seenivasan ◽

Santosh Kumar Kuncha ◽

Satya Brata Routh ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Active Site ◽

Unknown Function ◽

Physiological Importance ◽

Cellular Defense

Strict L-chiral rejection through Gly-cisPro motif during chiral proofreading underlies the inability of D-aminoacyl-tRNA deacylase (DTD) to discriminate between D-amino acids and achiral glycine. The consequent Gly-tRNAGly ‘misediting paradox’ is resolved by EF-Tu in the cell. Here, we show that DTD’s active site architecture can efficiently edit mischarged Gly-tRNAAla species four orders of magnitude more efficiently than even AlaRS, the only ubiquitous cellular checkpoint known for clearing the error. Also, DTD knockout in AlaRS editing-defective background causes pronounced toxicity in Escherichia coli even at low-glycine levels which is alleviated by alanine supplementation. We further demonstrate that DTD positively selects the universally invariant tRNAAla-specific G3•U70. Moreover, DTD’s activity on non-cognate Gly-tRNAAla is conserved across all bacteria and eukaryotes, suggesting DTD’s key cellular role as a glycine deacylator. Our study thus reveals a hitherto unknown function of DTD in cracking the universal mechanistic dilemma encountered by AlaRS, and its physiological importance.

Download Full-text