Arginine methylation is a post-translational modification found mostly in RNA-binding proteins. Poly(A)-binding protein II from calf thymus was shown by mass spectrometry and sequencing to contain N G ,N G -dimethylarginine at 13 positions in its amino acid sequence. Two additional arginine residues were partially methylated. Almost all of the modified residues were found in Arg-Xaa-Arg clusters in the C terminus of the protein. These motifs are distinct from Arg-Gly-Gly motifs that have been previously described as sites and specificity determinants for asymmetric arginine dimethylation. Poly(A)-binding protein II and deletion mutants expressed in Escherichia coli were in vitro substrates for two mammalian protein arginine methyltransferases, PRMT1 and PRMT3, with S-adenosyl-L-methionine as the methyl group donor. Both PRMT1 and PRMT3 specifically methylated arginines in the C-terminal domain corresponding to the naturally modified sites.

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(II1) 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 EID 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.Keywords: phosphomannose isomerase; Fe(II1)-dopamine complex ; zinc-dependent metalloenzyme ; EPR.Phosphomannose isomerase (PMI) catalyses the interconversion of fructose 6-phosphate and mannose 6-phosphate. The enzyme originally isolated from baker's yeast was shown to be a monomeric protein of molecular mass 45 kDa. It contains one zinc atodprotein monomer (Gracy and Noltmann, 1968 a). The enzyme catalyses the first committed step in the synthesis of cell wall mannoproteins from glycolytic intermediates. Temperaturesensitive mutations in the gene for PMI cause cell lysis and death at the restrictive temperature in both Saccharomyces cerevisiae (Smith et al., 1992) and Candida albicans (Smith et al., 1995). PMI is therefore a suitable target for the development of an inhibitory antifungal agent.The gene coding for PMI has been recently cloned from an ordered array genomic library of C. albicans cDNA (Smith et al., 1995) and overexpressed in Escherichia coli. Metal analysis of the recombinant protein showed the presence of almost one zinc ionIprotein monomer (Wells, T. N. C., unpublished results). However, at very high concentrations, for example greater than 50 mg/ml used in setting up crystallisation experiments (Tolley et al., 1994), the protein appeared blue. Comparison of the ultraviolet-visible range absorption spectrum with that of tyrosine hydroxylase Enzyme. Mannose-6-phosphate isomerase (phosphomannose isomerase) (EC 5.3.1.8).ion analysis showed the presence of 0.2 iron atomsIprotein molecule.Fermentation of E. coli under higher concentrations (1 mM) of FeSO, leads to the formation of a blue protein with a maximal iron concentration of 0.5 iron atomdprotein molecule. We have therefore analysed the protein and shown the blue colour to be ...