A Highly Active Protein Repair Enzyme from an Extreme Thermophile: Thel-Isoaspartyl Methyltransferase fromThermotoga maritima

Abstract

We show that the open reading frame in theThermotoga maritimagenome tentatively identified as thepcmgene (R. V. Swansonet al., J. Bacteriol.178, 484–489, 1996) does indeed encode a proteinl-isoaspartate (d-aspartate)O-methyltransferase (EC 2.1.1.77) and that this protein repair enzyme displays several novel features. We expressed the 317 amino acid pcm gene product of this thermophilic bacterium inEscherichia colias a fusion protein with an N-terminal 20 residue hexa-histidine-containing sequence. This protein contains a C-terminal domain of approximately 100 residues not previously seen in this enzyme from various prokaryotic or eukaryotic species and which does not have sequence similarity to any other entry in the GenBank databases. The C-terminal region appears to be required for enzymatic function as no activity is detected in two recombinant constructs lacking this domain. Sedimentation equilibrium analysis indicated that the enzyme is monomeric in solution. TheKmvalues for measured for peptide and protein substrates were found to be intermediate between those of the high-affinity human enzyme and those of the lower-affinity wheat, nematode, andE. colienzymes. The enzyme was extremely heat stable, with no loss of activity after 60 min at 100°C. Enzyme activity was observed at temperatures as high as 93°C with an optimal activity of 164 nmol/min/mg protein at 85°C. This activity is approximately 18-fold higher than the maximal activities of mesophilic homologs at 37°C. These data suggest that theThermotogaenzyme has unique features for initiating repair in damaged proteins containingl-isoaspartyl residues at elevated temperatures.