Identification of an Fe(III)-dihydroxyphenylalanine site in recombinant phosphomannose isomerase from Candida albicans.
Candida albicans phosphomannose isomerase (PMI) is a zinc metalloprotein of known crystal structure. When heterologously overexpressed in Escherichia coli, a blue protein that contains up to 0.5 iron atoms/PMI molecule could be isolated, with absorption maxima at 420 nm and 680 nm. These bands are reminiscent of ferric catecholate complexes, an assignment that has been confirmed by resonance Raman spectroscopy, and by reaction with Arnow's reagent, which is specific for the presence of 3,4-dihydroxyphenylalanine (Dopa). After enzymatic digestion of blue PMI, a peptide with the sequence DPHAXISG was isolated corresponding to residues Asp283-Gly290 in the amino acid sequence of C. albicans PMI, where the unidentified residue X287 is encoded by a tyrosine codon. It is proposed that iron and oxygen bring about hydroxylation of Tyr287 in PMI and that Fe(III) subsequently chelates the Dopa residue to give the characteristic absorption spectrum. The EPR spectrum of the blue protein suggests three iron environments in the protein, two in axial environments with E/D values approximately equal to 0.06 and 0.12 and one rhombic species. The nature of the iron co-ordination sites is discussed with the help of model systems and by comparison with other blue non-heme iron proteins.
Smith
J J
JJ
Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences, University of East Anglia, England.
Thomson
A J
AJ
Proudfoot
A E
AE
Wells
T N
TN
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics; D004295:Dihydroxyphenylalanine / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D007501:Iron / Q000378:metabolism*; D008359:Mannose-6-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry
D011994:Recombinant Proteins; D004295:Dihydroxyphenylalanine; D007501:Iron; D008359:Mannose-6-Phosphate Isomerase