Three distinct quinoprotein alcohol dehydrogenases are expressed when Pseudomonas putida is grown on different alcohols

Abstract

A bacterial strain that can utilize several kinds of alcohols as its sole carbon and energy sources was isolated from soil and tentatively identified as Pseudomonas putida HK5. Three distinct dye-linked alcohol dehydrogenases (ADHs), each of which contained the prosthetic group pyrroloquinoline quinone (PQQ), were formed in the soluble fractions of this strain grown on different alcohols. ADH I was formed most abundantly in the cells grown on ethanol and was similar to the quinoprotein ADH reported for P. putida (H. Görisch and M. Rupp, Antonie Leeuwenhoek 56:35-45, 1989) except for its isoelectric point. The other two ADHs, ADH IIB and ADH IIG, were formed separately in the cells grown on 1-butanol and 1,2-propanediol, respectively. Both of these enzymes contained heme c in addition to PQQ and functioned as quinohemoprotein dehydrogenases. Potassium ferricyanide was an available electron acceptor for ADHs IIB and IIG but not for ADH I. The molecular weights were estimated to be 69,000 for ADH IIB and 72,000 for ADH IIG, and both enzymes were shown to be monomers. Antibodies raised against each of the purified ADHs could distinguish the ADHs from one another. Immunoblot analysis showed that ADH I was detected in cells grown on each alcohol tested, but ethanol was the most effective inducer. ADH IIB was formed in the cells grown on alcohols of medium chain length and also on 1,3-butanediol. Induction of ADH IIG was restricted to 1,2-propanediol or glycerol, of which the former alcohol was more effective. These results from immunoblot analysis correlated well with the substrate specificities of the respective enzymes. Thus, three distinct quinoprotein ADHs were shown to be synthesized by a single bacterium under different growth conditions.

Roles for the Two 1-Butanol Dehydrogenases ofPseudomonas butanovorain Butane and 1-Butanol Metabolism

“…For type I quinoprotein ADH from P. aeruginosa ATCC 17933, the K m s for 2-butanol and S-(ϩ)-2-butanol were 680 and 980 M, respectively, while the K m s for the primary alcohols ethanol and 1-propanol were 14 and 21 M, respectively (28). For P. putida HK5, the K m s for ethanol and 1-butanol were 163 M and 1.62 mM, respectively (37).…”

“…In other oxidative nonmethylotrophic bacteria, ADHs have been classified into three groups (types I, II, and III) on the basis of their molecular properties, catalytic properties, and localization (22). The molecular structure of type I ADH found in Pseudomonas aeruginosa and Pseudomonas putida (15,16,37) resembles that of MDH but has very low affinity for methanol. Type I ADH uses a c-type cytochrome (cytochrome c QEDH or cytochrome c 550 ) as the electron acceptor (29,31), which subsequently reacts with another c-type cytochrome or coppercontaining protein (azurin) (4,26).…”

“…Type II ADHs are soluble periplasmic quinohemoproteins having PQQ and heme c as prosthetic groups and have been found in Comamonas testosteroni (12,17), P. putida (37), and Ralstonia eutropha (40). When P. putida HK5 is grown on ethanol, 1-butanol, and 1,2-propanediol, it produces three different quinoprotein ADHs: one type I ADH and two type II ADHs (ADH IIB and ADH IIG), respectively (37). Type III ADHs are membraneassociated enzymes found in the cytoplasmic membrane of acetic acid bacteria.…”

“…Type I ADH uses a c-type cytochrome (cytochrome c QEDH or cytochrome c 550 ) as the electron acceptor (29,31), which subsequently reacts with another c-type cytochrome or coppercontaining protein (azurin) (4,26). Type II ADHs are soluble periplasmic quinohemoproteins having PQQ and heme c as prosthetic groups and have been found in Comamonas testosteroni (12,17), P. putida (37), and Ralstonia eutropha (40). When P. putida HK5 is grown on ethanol, 1-butanol, and 1,2-propanediol, it produces three different quinoprotein ADHs: one type I ADH and two type II ADHs (ADH IIB and ADH IIG), respectively (37).…”

Molecular Cloning of Quinohemoprotein Alcohol Dehydrogenase, ADH IIB, fromPseudomonas putidaHK5

“…1) Recently the threedimensional structure of ADH IIB has been identied by X-ray crystallography. 2) In this study, the structural gene of ADH IIB was obtained by using PCR in vitro cloning techniques.…”