Maturity enzyme

As described earlier, translation of the EPSPS mRNA of plants results in the formation of a protein which has an AJ-terminal extension. The AJ-terminal extension, referred to as the chloroplast transit peptide, is necessary and sufficient for the import of the preprotein by the chloroplast. Once imported by the chloroplast, the transit peptide is cleaved releasing the mature enzyme. As expected, introduction of the EPSPS transit peptide to other protein sequences results in the importation of the fusion protein by the chloroplast. 

An enzyme in which the single catalytic residue is at the N-terminus of the protein. Many Ntn-hydrolases are synthesized as precursors and autoactivate the precursors are therefore peptidases, even if the mature enzyme has no further proteolytic activity. Three of the beta subunits of the proteasome are Ntn-hydrolases. 

These myeloperoxidase deficiencies may be hereditary or acquired. The genetics of hereditary myeloperoxidase deficiency are unknown, but a survey of patients by Nauseef and co-workers (Nauseef 1988) revealed (by im-munoblotting) that, in many patients, the mature enzyme was absent but promyeloperoxidase was present. Such observations suggest that in these patients the gene is normal and may be normally transcribed the defect resides in how this protein is processed and perhaps packaged. 

Formation of TPQ in the amine oxidases in which it occurs is initiated by binding of Cu to the precursor protein at the same site in which it functions as cofactor in the mature enzyme. Subsequently, the specific tyrosyl residue, the second amino acid in the conserved sequence Asn-Tyr-(Asp/Glu)-Tyr [44], is converted into TPQ in several steps, with the Cu catalyzing the oxidation of the precursor by 02 [45], Indications exist that this autocatalytic process is sometimes suboptimal most probably the insufficient availability of Cu and/or 02 in the posttranslational process is responsible [46],... 

Up to four cDNA clones have been isolated for potato BE - one for 91 to 99 kDa.253-255 All these allelic clones have sequences similar to those of the BEI type. Also, the sbelc allele codes for a mature enzyme of 830 amino acids with a MW of 95.18 kDa. The sbelc BE protein product, expressed in E. coli, migrates as a 103 kDa protein.253 It is of interest to note that BE isolated from other plants, bacteria and mammals have molecular masses ranging from 75 to —85 kDa. These molecular weights are consistent with the molecular weights obtained from deduced amino acid sequences obtained from isolated genes or cDNA clones. 

The coding region of the type-L isozyme is 2898 base pairs long. It corresponds to 966 amino acid residues with a molecular weight of 109,648, and consists of two regions the amino-terminal extended sequence of 50 residues and the mature protein sequence of 916 residues. The amino acid sequence of the mature protein deduced from the cDNA structure is perfectly matched to the sequence chemically determined as described above. The stop codon, TAA, appeared at position 2942 in the cDNA sequence just after the carboxyl-terminal amino acid of the mature enzyme, indicating that no post-translational processing occurred at the carboxyl terminus. 

Mutant m-51 also contained three substitutions AlaBBOVal, Ala98l=>Thr in the mature enzyme and AlaSl Thr in the pro-region. The suppressor mutation was found to be Ala31 OThr. None of the cold active variants in this study showed any loss of stability relative to wild type. This work demonstrates that activity can be increased at low temperatures, and that it can be done at no cost to thermostability. 

The simple coordination chemistry characteristic of the majority of protein-metal interactions is replaced in certain cases by irreversible covalent modifications of the protein mediated by the metal ion. These modifications are essential for the function and are templated by the structure of the protein, as no other proteins are required for the reaction to occur. These self-processing reactions result in the biogenesis of redox cofactors in some enzymes (amine oxidases, galactose oxidase, cytochrome c oxidase) and activation of hydrolytic sites in others (nitrile hydratase). The active sites of all of these enzymes are bifunctional, directing not only the catalytic turnover reaction of the mature enzyme but the modification steps required for maturation. 

The recombinant C3 exoenzyme that we used is a modified enzyme that does not contain the signal peptide, but has Met-Ala attached to Ser of the mature enzyme (Kumagai et a/., 1993). The modified gene was cloned into pET 3a vector, resulting in pET 3a C3, which allows the enzyme to be expressed under control of the bacteriophage T7 promoter. E. coli strain BL21 (DE3) cells are transformed with pET 3a C3. 

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