It has been assumed that CODHII Ch catalyzes the oxidation of CO at the Ni-( 2 S)-Fe1 subsite of cluster C (3). The prime candidate for CO binding is the nickel ion because of its facile accessibility through the substrate channel and its empty apical coordination site (3). Fe1 is the presumed OH Ϫ donor ligand in CO 2 formation (3, 6). The CODH Oc from the aerobic bacterium Oligotropha carboxidovorans oxidizes CO at the Mo-( 2 S)-Cu subsite of the [Cu-S-MoO 2 ] active site, in which copper and molybdenum are bridged by a cyanolyzable sulfane 2 S (7, 8). The enzyme is inactivated when 2 S is removed and reactivated when 2 S is reinserted (9). The Mo-( 2 S)-Cu subsite resembles the Ni-( 2 S)-Fe1 bridge in cluster C of CODHII Ch . The mechanism of CO oxidation based on the x-ray structure of [Cu-S-MoO 2 ] CODH Oc with bound inhibitor n-butyl isocyanide involves a thiocarbonate-like intermediate state and proposes the binding of CO between the 2 S and copper (equivalent to nickel in CODHII Ch ) and the binding of an OH Ϫ group at molybdenum (equivalent to Fe1 in CODHII Ch ) (7).Structures of cluster C of Ni-Fe CODHs from Rhodospirillum rubrum (CODH Rr ) (10) and Moorella thermoacetica (CODH Mt ) (11, 12) also showed the positions of the five metal ions in cluster C of CODHII Ch but did not reveal the bridging 2 S. Since sodium sulfide was found to inhibit CODH Rr and CODH Mt , it has been concluded that cluster C with the bridging 2 S, as has been observed in CODHII Ch from C. hydrogenoformans (3, 4), might represent an inhibited form (13). On the other hand, it has been shown that the [Ni-4Fe-4S] cluster miss-

In addition to the known response regulator ErbR (former AgmR) and the two-component regulatory system EraSR (former ExaDE), three additional regulatory proteins have been identified as being involved in controlling transcription of the aerobic ethanol oxidation system in Pseudomonas aeruginosa. Two putative sensor kinases, ErcS and ErcS9, and a response regulator, ErdR, were found, all of which show significant similarity to the two-component flhSR system that controls methanol and formaldehyde metabolism in Paracoccus denitrificans. All three identified response regulators, EraR (formerly ExaE), ErbR (formerly AgmR) and ErdR, are members of the luxR family. The three sensor kinases EraS (formerly ExaD), ErcS and ErcS9 do not contain a membrane domain. Apparently, they are localized in the cytoplasm and recognize cytoplasmic signals. Inactivation of gene ercS caused an extended lag phase on ethanol. Inactivation of both genes, ercS and ercS9, resulted in no growth at all on ethanol, as did inactivation of erdR. Of the three sensor kinases and three response regulators identified thus far, only the EraSR (formerly ExaDE) system forms a corresponding kinase/regulator pair. Using reporter gene constructs of all identified regulatory genes in different mutants allowed the hierarchy of a hypothetical complex regulatory network to be established. Probably, two additional sensor kinases and two additional response regulators, which are hidden among the numerous regulatory genes annotated in the genome of P. aeruginosa, remain to be identified.

Gluconobacter oxydans converts glucose to gluconic acid and subsequently to 2-keto-D-gluconic acid (2-KGA) and 5-keto-D-gluconic acid (5-KGA) by membrane-bound periplasmic pyrroloquinoline quinone-dependent and flavin-dependent dehydrogenases. The product pattern obtained with several strains differed significantly. To increase the production of 5-KGA, which can be converted to industrially important L-(+)-tartaric acid, growth parameters were optimized. Whereas resting cells of G. oxydans ATCC 621H converted about 11% of the available glucose to 2-KGA and 6% to 5-KGA, with growing cells and improved growth under defined conditions (pH 5, 10% pO2, 0.05% pCO2) a conversion yield of about 45% 5-KGA from the available glucose was achieved. As the accumulation of the by-product 2-KGA is highly disadvantageous for an industrial application of G. oxydans, a mutant was generated in which the membrane-bound gluconate-2-dehydrogenase complex was inactivated. This mutant, MF1, grew in a similar way to the wild type, but formation of the undesired 2-KGA was not observed. Under improved growth conditions, mutant MF1 converted the available glucose almost completely (84%) into 5-KGA. Therefore, this newly developed recombinant strain is suitable for the industrial production of 5-KGA.

Gluconobacter oxydans DSM 2343 (ATCC 621H)catalyzes the oxidation of glucose to gluconic acid and subsequently to 5-keto-D-gluconic acid (5-KGA), a precursor of the industrially important L-(+)-tartaric acid. To further increase 5-KGA production in G. oxydans, the mutant strain MF1 was used. In this strain the membrane-bound gluconate-2-dehydrogenase activity, responsible for formation of the undesired by-product 2-keto-D-gluconic acid, is disrupted. Therefore, high amounts of 5-KGA accumulate in the culture medium. G. oxydans MF1 was equipped with plasmids allowing the overexpression of the membrane-bound enzymes involved in 5-KGA formation. Overexpression was confirmed on the transcript and enzymatic level. Furthermore, the resulting strains overproducing the membrane-bound glucose dehydrogenase showed an increased gluconic acid formation, whereas the overproduction of gluconate-5-dehydrogenase resulted in an increase in 5-KGA of up to 230 mM. Therefore, these newly developed recombinant strains provide a basis for further improving the biotransformation process for 5-KGA production.