Glutamic acid-141: a heme 'bodyguard' in anionic tobacco peroxidase.
The role of the conserved glutamic acid residue in anionic plant peroxidases with regard to substrate specificity and stability was examined. A Glu141Phe substitution was generated in tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases such as horseradish peroxidase C (HRP C). The newly constructed enzyme was compared to wild-type recombinant TOP and HRP C expressed in E. coli. The Glu141Phe substitution supports heme entrapment during the refolding procedure and increases the reactivation yield to 30% compared to 7% for wild-type TOP. The mutation reduces the activity towards ABTS, o-phenylenediamine, guaiacol and ferrocyanide to 50% of the wild-type activity. No changes are observed with respect to activity for the lignin precursor substrates, coumaric and ferulic acid. The Glu141Phe mutation destabilizes the enzyme upon storage and against radical inactivation, mimicking inactivation in the reaction course. Structural alignment shows that Glu141 in TOP is likely to be hydrogen-bonded to Gln149, similar to the Glu143-Lys151 bond in Arabidopsis A2 peroxidase. Supposedly, the Glu141-Gln149 bond provides TOP with two different modes of stabilization: (1) it prevents heme dissociation, i.e., it 'guards' heme inside the active center; and (2) it constitutes a shield to protect the active center from solvent-derived radicals.
Hushpulian
Dmitri M
DM
Department of Chemical Enzymology, Chemical Faculty, M.V. Lomonosov Moscow State University, Moscow 119992, Russia. hushpulian@gmail.com
Poloznikov
Andrew A
AA
Savitski
Pavel A
PA
Rozhkova
Alexandra M
AM
Chubar
Tatyana A
TA
Fechina
Victoria A
VA
Orlova
Marina A
MA
Tishkov
Vladimir I
VI
Gazaryan
Irina G
IG
Lagrimini
L Mark
LM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D052160:Benzothiazoles / Q000378:metabolism; D003962:Dianisidine / Q000378:metabolism; D005720:Gamma Rays; D018698:Glutamic Acid / Q000737:chemistry*; D006139:Guaiacol / Q000378:metabolism; D006418:Heme / Q000737:chemistry*; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D017510:Protein Folding; D011994:Recombinant Proteins / Q000302:isolation & purification; D013379:Substrate Specificity; D013451:Sulfonic Acids / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology
D052160:Benzothiazoles; D011994:Recombinant Proteins; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D018698:Glutamic Acid; D006418:Heme; D006139:Guaiacol; D010544:Peroxidases; C085547:tobacco anionic peroxidase; D003962:Dianisidine