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45. LolA-dependent release of a lipid-modified protein from the inner membrane of Escherichia coli requires nucleoside triphosphate.

Author

Yakushi, T, Yokota, N, Matsuyama, S, and Tokuda, H

Abstract

The outer membrane-directed lipoproteins are released from the inner membrane of Escherichia coli as a complex with LolA, a periplasmic chaperone. The LolA-dependent release of lipoproteins is critical for lipoprotein sorting as it depends on the outer membrane-specific sorting signal. To clarify molecular events involved in the LolA-dependent lipoprotein release, we attempted to establish an in vitro assay system. The major outer membrane lipoprotein (Lpp) was found to lose its release competence soon after being processed to mature Lpp in the inner membrane and therefore could not be used as a substrate for an in vitro system. An Lpp derivative, L10P, was constructed and found to retain the release competence long after its maturation. L10P was synthesized and radiolabeled in spheroplasts in the absence of LolA; therefore, it remained anchored to the inner membrane of spheroplasts. Right-side out membrane vesicles containing L10P were then prepared and used to examine the release of L10P. In addition to LolA, L10P release absolutely required nucleoside triphosphate (NTP). A non-hydrolyzable NTP analogue strongly inhibited the NTP-dependent release. The outer membrane-specific sorting signal was essential for the in vitro release of L10P. Furthermore, L10P released in vitro was specifically incorporated into the outer membrane. These results indicate that the in vitro release of L10P represents an in vivo reaction and requires energy.

Published
1998

46. Function of multiple heme c moieties in intramolecular electron transport and ubiquinone reduction in the quinohemoprotein alcohol dehydrogenase-cytochrome c complex of Gluconobacter suboxydans.

Author

Matsushita, K, Yakushi, T, Toyama, H, Shinagawa, E, and Adachi, O

Abstract

Alcohol dehydrogenase (ADH) of acetic acid bacteria functions as the primary dehydrogenase of the ethanol oxidase respiratory chain, where it donates electrons to ubiquinone. ADH is a membrane-bound quinohemoprotein-cytochrome c complex which consists of subunits I (78 kDa), II (48 kDa), and III (14 kDa) and contains several hemes c as well as pyrroloquinoline quinone as prosthetic groups. To understand the role of the heme c moieties in the intramolecular electron transport and the ubiquinone reduction, the ADH complex of Gluconobacter suboxydans was separated into a subunit I/III complex and subunit II, then reconstituted into the complex. The subunit I/III complex, probably subunit I, contained 1 mol each of pyrroloquinoline quinone and heme c and exhibited significant ferricyanide reductase, but no Q1 reductase activities. Subunit II was a triheme cytochrome c and had no enzyme activity, but it enabled the subunit I/III complex to reproduce the Q1 and ferricyanide reductase activities. Hybrid ADH consisting of the subunit I/III complex of G. suboxydans ADH and subunit II of Acetobacter aceti ADH was constructed and it had showed a significant Q1 reductase activity, indicating that subunit II has a ubiquinone-binding site. Inactive ADH from G. suboxydans exhibiting only 10% of the Q1 and ferricyanide reductase activities of the active enzyme has been isolated separately from active ADH (Matsushita, K., Yakushi, T., Takaki, Y., Toyama, H., and Adachi, O (1995) J. Bacteriol. 177, 6552-6559). Using these active and inactive ADHs and also isolated subunit I/III complex, we performed kinetic studies which suggested that ADH contains four ferricyanide-reacting sites, one of which was detected in subunit I and the others in subunit II. One of the three ferricyanide-reacting sites in subunit II was defective in inactive ADH. The ferricyanide-reacting site remained inactive even after alkali treatment of inactive ADH and also after reconstituting the ADH complex from the subunits, in contrast to the restoration of Q1 reductase activity and the other ferricyanide reductase activities. Thus, the data suggested that the heme c in subunit I and two of the three heme c moieties in subunit II are involved in the intramolecular electron transport of ADH into ubiquinone, where one of the two heme c sites may work at, or close to, the ubiquinone-reacting site and another between that and the heme c site in subunit I. The remaining heme c moiety in subunit II may have a function other than the electron transfer from ethanol to ubiquinone in ADH.

Published
1996

49. DETERMINATION OF DEHYDROGENASE ACTIVITY IN GLUCONOBACTER AND ACETOBACTER STRAINS.

Author

Sainz, F., Torija, M. J., Mas, A., Matsutani, M., Kataoka, N., Yakushi, T., and Matsushita, K.

Subjects
DEHYDROGENASES, ACETOBACTER, ORGANIC acids
Abstract

The acetic acid bacteria (AAB) are known for the ability to perform quick and incomplete oxidations of a high number of alcohols and carbohydrates, resulting in the accumulation of organic acids as final products. Sugar and sugar alcohols are the substrates preferred by Gluconobacter species, while Acetobacter species show higher preference for ethanol. Examples of this oxidation activity are the production of acetic acid from ethanol or D-gluconic acid and derivatives from glucose. Two different groups of enzymes, which differ in the localization and the substrate specificity, are responsible to carry out D-glucose oxidation. The main pathway is the one linked to the bacterial membrane, where D-glucose is directly oxidized to D-gluconic acid by membrane-bound pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (mGDH). This acid could be further oxidized to 2-keto-D-gluconate (2-KGA) and 2,5-diketo-Dgluconate by a flavin-dependent gluconate-2-dehydrogenase (GA2DH) and 2-keto-D-gluconate dehydrogenase (2KGDH), respectively. Additionally, D-gluconate could also be oxidized to 5-ketogluconic acid (5-KGA) by a membrane-bound PQQ-dependent glycerol dehydrogenase which could be called as gluconate-5-dehydrogenase (GA5DH) by focusing on gluconate metabolism. The aim of this work was to determine the enzymatic activity of these four membrane- bound enzymes in 3 AAB strains isolated from strawberry and grape, compared with each type strains. Thus six strains of AAB (2 strains of Gluconobacter oxydans, 2 of Gluconobacter japonicus and 2 of Acetobacter malorum) were grown in synthetic medium with 30 g/L of D-glucose and 40 g/L of fructose at different growth phases (24, 48 and 96 h). In general, strains of Gluconobacter genus presented higher activity in these enzymes than the A. malorum strains. In Gluconobacter strains, the activity of mGDH and GA2DH was extremely high and reasonable, respectively, while in A. malorum strains, reasonable mGDH activity and low but significant GA2DH activity were detected. Additionally, G. oxydans strains were the only ones that showed a reasonable GA5DH activity while G. japonicus and A. malorum presented a low and very low enzyme activity. Finally, none of the strains showed 2KGDH activity. [ABSTRACT FROM AUTHOR]

Published
2015

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