Site Directed natural ones

The single-crystal nature of the majority of bacterial magnetites implies that nucleation of magnetite from the iron(III) precursor phase occurs at one primary nucleation site that grows at the expense of other potential sites. It is probable, therefore, that the surrounding magnetosome membrane plays a crucial role in the generation of a local environment for site-directed nucleation. One possibility is that... 

The geometric nature of the problem is posed by the very structure of a carboxylic acid 4. If two, or even more, of such functions are to converge on a single basic site, the least one can anticipate is that a U-turn must be engineered into the system. This arises from the direction of the carbonyl-C, bond which points in a direction roughly opposite to the direction indicated by the OH bond. From this perspective, the tricarboxylic acid 5 a first described by Kemp 8) offers an unusually favorable architecture. In this, three methyls force the smaller carboxyl groups to assume a triaxial... 

Chemical modification of surface residues of HRP is one method which may offer some improvement in thermal or long-term stability of the enzyme. The -amino groups of the six surface Lys residues can be modified by reaction with carboxylic anhydrides and picryl sulfonic acid (296). In this example the number of sites modified was found to be more significant than the chemical nature of the modification, at least as a criterion for improved stability. Other methods explored include the use of bifunctional crosslinking reagents to couple surface sites on the enzyme (297). Future developments are likely to be concerned with the selection of site-directed mutants of HRP C that show enhanced thermal stability. Dramatic increases in thermal stability of up to 190-fold have been reported recently for mutants of Coprinus cinereus peroxidase (CIP) generated using a directed evolution approach (298). 

Unstable proteins may be modified by the molecular biological technique of site-directed mutagenesis to remove the site of instability— for instance, an oxidizable cysteine. Such techniques are appropriate for commercial production of proteins, but may of course alter natural functioning parameters. Increased thermostability can be one modification, although it is not easy to predict mutations that will improve that parameter. Thermostable proteins originating from thermophilic bacteria do not need structural modification and, if expressed in large amounts, can be purified satisfactorily in one step by simply heat-treating the extract at 70°C for 30 min, which denatures virtually all the host proteins (e.g., see Oka et al., 1989). 

One set of site-directed mutagenesis studies improved the peroxygenase activity of Mb [128] in a different random mutagenesis study the peroxidase activity was improved. Clearly these reactions are very different from one another, and neither is myoglobin s natural function. Here is another example where an alternative scaffold supports functions that are characteristic of other conserved structures. Heme enzyme function is flexible and highly evolvable. 

The change of the acceptor affinity of a GT was demonstrated by the group of X. Wang. The catal3dic efficiency of the GT UGT85H2 from Medicago tmncatula towards the natural acceptor kaempferol (20) was increased 37-fold based on only one amino acid substitution. Another substitution dramatically improved (54-fold) the turnover rate and catalytic efficiency for biochanin A (21), the second natural acceptor (79). This work impressively presents how site-directed mutagenesis could lead to radical improvement of the catalytic efficiency. 

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