Repair deficient mice reveal mABH2 as the primary oxidative demethylase for repairing 1meA and 3meC lesions in DNA.

Two human homologs of the Escherichia coli AlkB protein, denoted hABH2 and hABH3, were recently shown to directly reverse 1-methyladenine (1meA) and 3-methylcytosine (3meC) damages in DNA. We demonstrate that mice lacking functional mABH2 or mABH3 genes, or both, are viable and without overt phenotypes. Neither were histopathological changes observed in the gene-targeted mice. However, in the absence of any exogenous exposure to methylating agents, mice lacking mABH2, but not mABH3 defective mice, accumulate significant levels of 1meA in the genome, suggesting the presence of a biologically relevant endogenous source of methylating agent. Furthermore, embryonal fibroblasts from mABH2-deficient mice are unable to remove methyl methane sulfate (MMS)-induced 1meA from genomic DNA and display increased cytotoxicity after MMS exposure. In agreement with these results, we found that in vitro repair of 1meA and 3meC in double-stranded DNA by nuclear extracts depended primarily, if not solely, on mABH2. Our data suggest that mABH2 and mABH3 have different roles in the defense against alkylating agents.

Rational engineering of enzyme stability.

During the past 15 years there has been a continuous flow of reports describing proteins stabilized by the introduction of mutations. These reports span a period from pioneering rational design work on small enzymes such as T4 lysozyme and barnase to protein design, and directed evolution. Concomitantly, the purification and characterization of naturally occurring hyperstable proteins has added to our understanding of protein stability. Along the way, many strategies for rational protein stabilization have been proposed, some of which (e.g. entropic stabilization by introduction of prolines or disulfide bridges) have reasonable success rates. On the other hand, comparative studies and efforts in directed evolution have revealed that there are many mutational strategies that lead to high stability, some of which are not easy to define and rationalize. Recent developments in the field include increasing awareness of the importance of the protein surface for stability, as well as the notion that normally a very limited number of mutations can yield a large increase in stability. Another development concerns the notion that there is a fundamental difference between the "laboratory stability" of small pure proteins that unfold reversibly and completely at high temperatures and "industrial stability", which is usually governed by partial unfolding processes followed by some kind of irreversible inactivation process (e.g. aggregation). Provided that one has sufficient knowledge of the mechanism of thermal inactivation, successful and efficient rational stabilization of enzymes can be achieved.

Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface.

The stability of tetrameric malate dehydrogenase from the green phototrophic bacterium Chloroflexus aurantiacus (CaMDH) is at least in part determined by electrostatic interactions at the dimer-dimer interface. Since previous studies had indicated that the thermal stability of CaMDH becomes lower with increasing pH, attempts were made to increase the stability by removal of (excess) negative charge at the dimer-dimer interface. Mutation of Glu165 to Gln or Lys yielded a dramatic increase in thermal stability at pH 7.5 (+23.6 -- + 23.9 degrees C increase in apparent t(m)) and a more moderate increase at pH 4.4 (+4.6 -- + 5.4 degrees C). The drastically increased stability at neutral pH was achieved without forfeiture of catalytic performance at low temperatures. The crystal structures of the two mutants showed only minor structural changes close to the mutated residues, and indicated that the observed stability effects are solely due to subtle changes in the complex network of electrostatic interactions in the dimer-dimer interface. Both mutations reduced the concentration dependency of thermal stability, suggesting that the oligomeric structure had been reinforced. Interestingly, the two mutations had similar effects on stability, despite the charge difference between the introduced side-chains. Together with the loss of concentration dependency, this may indicate that both E165Q and E165K stabilize CaMDH to such an extent that disruption of the inter-dimer electrostatic network around residue 165 no longer limits kinetic thermal stability.

Stabilization of a tetrameric malate dehydrogenase by introduction of a disulfide bridge at the dimer-dimer interface.

Malate dehydrogenase (MDH) from the moderately thermophilic bacterium Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from mesophilic organisms usually are dimers. To investigate the potential contribution of the extra dimer-dimer interface in CaMDH with respect to thermal stability, we have engineered an intersubunit disulfide bridge designed to strengthen dimer-dimer interactions. The resulting mutant (T187C, containing two 187-187 disulfide bridges in the tetramer) showed a 200-fold increase in half-life at 75 degrees C and an increase of 15 deg. C in apparent melting temperature compared to the wild-type. The crystal structure of the mutant (solved at 1.75 A resolution) was essentially identical with that of the wild-type, with the exception of the added inter-dimer disulfide bridge and the loss of an aromatic intra-dimer contact. Remarkably, the mutant and the wild-type had similar temperature optima and activities at their temperature optima, thus providing a clear case of uncoupling of thermal stability and thermoactivity. The results show that tetramerization may contribute to MDH stability to an extent that depends strongly on the number of stabilizing interactions in the dimer-dimer interface.

Electrostatic interactions across the dimer-dimer interface contribute to the pH-dependent stability of a tetrameric malate dehydrogenase.

Malate dehydrogenase (MDH) from the moderately thermophilic bacterium Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from mesophilic bacteria usually are dimers. Using site-directed mutagenesis, we show here that a network of electrostatic interactions across the extra dimer-dimer interface in CaMDH is important for thermal stability and oligomeric integrity. Stability effects of single point mutations (E25Q, E25K, D56N, D56K) varied from -1.2 degrees C to -26.8 degrees C, and depended strongly on pH. Gel-filtration experiments indicated that the 26.8 degrees C loss in stability observed for the D56K mutant at low pH was accompanied by a shift towards a lower oligomerization state.

Structural basis for thermophilic protein stability: structures of thermophilic and mesophilic malate dehydrogenases.

The three-dimensional structure of four malate dehydrogenases (MDH) from thermophilic and mesophilic phototropic bacteria have been determined by X-ray crystallography and the corresponding structures compared. In contrast to the dimeric quaternary structure of most MDHs, these MDHs are tetramers and are structurally related to tetrameric malate dehydrogenases from Archaea and to lactate dehydrogenases. The tetramers are dimers of dimers, where the structures of each subunit and the dimers are similar to the dimeric malate dehydrogenases. The difference in optimal growth temperature of the corresponding organisms is relatively small, ranging from 32 to 55 degrees C. Nevertheless, on the basis of the four crystal structures, a number of factors that are likely to contribute to the relative thermostability in the present series have been identified. It appears from the results obtained, that the difference in thermostability between MDH from the mesophilic Chlorobium vibrioforme on one hand and from the moderate thermophile Chlorobium tepidum on the other hand is mainly due to the presence of polar residues that form additional hydrogen bonds within each subunit. Furthermore, for the even more thermostable Chloroflexus aurantiacus MDH, the use of charged residues to form additional ionic interactions across the dimer-dimer interface is favored. This enzyme has a favorable intercalation of His-Trp as well as additional aromatic contacts at the monomer-monomer interface in each dimer. A structural alignment of tetrameric and dimeric prokaryotic MDHs reveal that structural elements that differ among dimeric and tetrameric MDHs are located in a few loop regions.