Spectroscopic studies on an oxygen-binding haemoglobin-like flavohaemoprotein from Escherichia coli

The Escherichia coli haemoglobin-like flavohaemoprotein (Hmp) has been purified to near homogeneity using two chromatographic steps. The prosthetic groups are identified as FAD and protohaem IX. SDS/PAGE has indicated a molecular mass of 44 kDa for the monomeric protein consistent with the amino-acid sequence deduced from the hmp+ gene. The protein, as isolated, is in the Fe(III) state, exhibiting absorbance maxima at 403.5, 540 (shoulder) and 627 nm. The ferrous and carbonmonoxyferrous states resemble those of haemoglobin, showing maxima at 431.5 and 558 nm, and 421, 542 and 566 nm respectively. Upon aerobic addition of NAD(P)H, the ferric state is reduced to the oxygenated Fe(II) state, characterized by maxima at 413, 544 and 580 nm. This oxy form is not stable and slowly decays to the ferric state. Addition of dithionite and nitrite to the ferric protein results in the formation of a nitrosyl complex, whose e.p.r. characteristics indicate that the b-type haem is attached to the protein through a nitrogenous ligand, probably originating from a histidine residue.

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The amino acid sequence encompassing the active site histidine residue of lipoamide dehydrogenase from Escherichia coli labelled with a bifunctional arsenoxide

Flavins and Flavoproteins ◽

10.1515/9783111521350-027 ◽

1984 ◽

pp. 157-160

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

Active Site ◽

Histidine Residue ◽

Lipoamide Dehydrogenase ◽

Active Site Histidine

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Triphenylmethane Reductase from Citrobacter sp. Strain KCTC 18061P: Purification, Characterization, Gene Cloning, and Overexpression of a Functional Protein in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.7955-7960.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 7955-7960 ◽

Cited By ~ 53

Author(s):

Moon-Sun Jang ◽

Young-Mi Lee ◽

Cheorl-Ho Kim ◽

Jai-Heon Lee ◽

Dong-Woo Kang ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

Molecular Mass ◽

Enzymatic Reaction ◽

Amino Acid Sequences ◽

Binding Motif ◽

Triphenylmethane Dyes ◽

Functional Protein ◽

Triphenylmethane Reductase

ABSTRACT We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60°C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.

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Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646610 ◽

1989 ◽

Vol 61 (03) ◽

pp. 437-441 ◽

Cited By ~ 28

Author(s):

Cindra Condra ◽

Elka Nutt ◽

Christopher J Petroski ◽

Ellen Simpson ◽

P A Friedman ◽

...

Keyword(s):

Molecular Weight ◽

Salivary Gland ◽

Amino Acid ◽

Amino Acid Sequence ◽

Western Blot ◽

Potent Inhibitor ◽

Factor Xa ◽

Coagulation Factor ◽

Sds Page ◽

Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

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