Tyrosine-728 and glutamic acid-735 are essential for the metalloproteolytic activity of the lethal factor of Bacillus anthracis.
The lethal factor (LF) of Bacillus anthracis is a Zn2+-endopeptidase specific for the MAPK-kinase family of proteins. The catalytic zinc atom is coordinated by a first shell of residues including the two histidines and the glutamate of the zinc-binding motif HExxH and by Glu-735. A characteristic feature of LF is the presence, within the second shell of residues, of a tyrosine (Tyr-728) in close proximity (3.3 A) to the zinc atom. To investigate the role of Tyr-728 and Glu-735, LF mutants with one or both of these two residues replaced by Ala were cloned, expressed, and purified from Escherichia coli. A fourth mutant was obtained by replacing Tyr-728 with Phe. Spectroscopic analysis of these mutants indicates that they fold in the same way as the parental molecule and that zinc stabilizes the structure of LF. These mutants have neither proteolytic activity nor in vivo toxicity. The possible role of Tyr-728 in catalysis is discussed.
Tonello
Fiorella
F
Istituto di Neuroscienze del CNR, Università di Padova, Via G Colombo 3, 35121 Padua, Italy. fiorella.tonello@unipd.it
Naletto
Laura
L
Romanello
Vanina
V
Dal Molin
Federica
F
Montecucco
Cesare
C
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000378:metabolism*; D001427:Bacterial Toxins; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D008264:Macrophages / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D051381:Rats; D011916:Rats, Inbred F344; D011994:Recombinant Proteins / Q000737:chemistry; D013053:Spectrophotometry; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry*; D014508:Urea / Q000494:pharmacology; D015032:Zinc / Q000737:chemistry
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D002352:Carrier Proteins; D011994:Recombinant Proteins; C030325:anthrax toxin; D018698:Glutamic Acid; D014443:Tyrosine; D006639:Histidine; D014508:Urea; D005982:Glutathione Transferase; D020929:Mitogen-Activated Protein Kinase Kinases; D015032:Zinc