Histidine mutations

Aside from the expression of histidine mutations that are easily detected, other properties have been built into the Salmonella strains by mutation to increase their sensitivity. The strains cure defective in DNA excision repair (uvrB). In this case, the increased sensitivity probably is due to the failure to remove some DNA adducts that could lead to mutation. The strains also possess a mutation (rfa) that removes part of the lipopolysaccharide barrier of the bacterial cell wall and thereby makes the cells more permeable to some chemicals. Finally, Salmonella strains TA98 and TA100 contain the R-factor plasmid pkMIOl,277 which increases sensitivity probably by increasing the activity of an error-prone DNA-repair system. 

Oya, Y. and Yamamoto, K. (1988) The biological activity of hydrogen peroxide. IV. Enhancement of its clastogenic actions by coadministration of L-histidine. Mutat. Res. 198 ... 

Fio. 3. Genetic map of histidine mutations in Salmonella. The genes specifying the histidine enzymes, hisA through hisi, are in a cluster near 6 o clock on the map. HisR, U, W, S, O, and T are regulatory genes (see Section IV). HisP is involved with histidine trans port. This map is adapted from Fink and Roth [42]. 

The results of experiments in which the mutation was made were, however, a complete surprise. The Asp 189-Lys mutant was totally inactive with both Asp and Glu substrates. It was, as expected, also inactive toward Lys and Arg substrates. The mutant was, however, catalytically active with Phe and Tyr substrates, with the same low turnover number as wild-type trypsin. On the other hand, it showed a more than 5000-fold increase in kcat/f m with Leu substrates over wild type. The three-dimensional structure of this interesting mutant has not yet been determined, but the structure of a related mutant Asp 189-His shows the histidine side chain in an unexpected position, buried inside the protein. 

In principle, numerous reports have detailed the possibility to modify an enzyme to carry out a different type of reaction than that of its attributed function, and the possibility to modify the cofactor of the enzyme has been well explored [8,10]. Recently, the possibility to directly observe reactions, normally not catalyzed by an enzyme when choosing a modified substrate, has been reported under the concept of catalytic promiscuity [9], a phenomenon that is believed to be involved in the appearance of new enzyme functions during the course of evolution [23]. A recent example of catalytic promiscuity of possible interest for novel biotransformations concerns the discovery that mutation of the nucleophilic serine residue in the active site of Candida antarctica lipase B produces a mutant (SerlOSAla) capable of efficiently catalyzing the Michael addition of acetyl acetone to methyl vinyl ketone [24]. The oxyanion hole is believed to be complex and activate the carbonyl group of the electrophile, while the histidine nucleophile takes care of generating the acetyl acetonate anion by deprotonation of the carbon (Figure 3.5). 

Early mutational studies of the Rieske protein from 6ci complexes have been performed with the intention of identifying the ligands of the Rieske cluster. These studies have shown that the four conserved cysteine residues as well as the two conserved histidine residues are essential for the insertion of the [2Fe-2S] cluster (44, 45). Small amounts of a Rieske cluster with altered properties were obtained in Rhodobacter capsulatus when the second cysteine in the cluster binding loop II (Cys 155, corresponding to Cys 160 in the bovine ISF) was replaced by serine (45). The fact that all four cysteine residues are essential in Rieske clusters from be complexes, but that only two cysteines are conserved in Rieske-type clusters, led to the suggestion that the Rieske protein may contain a disulfide bridge the disulfide bridge was finally shown to exist in the X-ray structure (9). 

Santisteban, I., Arredondo-Vega, F. X., Kelly, S., Debre, M., Fischer, A., Pdrignon, J. L., Hilman, B., Eldahr, J., Dreyfus, D. H., Howell, P. L., and Hershfield, M. S Four new adenosine deaminase mutations, altering a zinc-binding histidine, two conserved alanine, and a 5 splice site. Hum. Mutat. 5,243-250 (1995). 

Figure 4.2 Hypothetical plasma membrane (PM)-associated structure of FR02. Four histidine residues (white spots) predicted to coordinate two intramembraneous haem groups (white bars) are indicated, as are the tetrapeptide binding sites for FAD and N AD(P)H. The sites of mutations in the FRO gene are indicated (frdl-l,frdl-3) i, inside cell o, outside cell. Reprinted with permission from Nature (Robinson et al., 1999). Copyright (1999) Macmillan Magazines Limited.

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