Publications

• Functional Role of Lanthanides in Enzymatic Activity and Transcriptional Regulation of Pyrroloquinoline Quinone-Dependent Alcohol Dehydrogenases in Pseudomonas putida KT2440.

  Wehrmann M., Billard P., Martin-Meriadec A., Zegeye A., Klebensberger J.

  The oxidation of alcohols and aldehydes is crucial for detoxification and efficient catabolism of various volatile organic compounds (VOCs). Thus, many Gram-negative bacteria have evolved periplasmic oxidation systems based on pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) th ... >> More

  The oxidation of alcohols and aldehydes is crucial for detoxification and efficient catabolism of various volatile organic compounds (VOCs). Thus, many Gram-negative bacteria have evolved periplasmic oxidation systems based on pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) that are often functionally redundant. Here we report the first description and characterization of a lanthanide-dependent PQQ-ADH (PedH) in a nonmethylotrophic bacterium based on the use of purified enzymes from the soil-dwelling model organism <i>Pseudomonas putida</i> KT2440. PedH (PP_2679) exhibits enzyme activity on a range of substrates similar to that of its Ca²⁺-dependent counterpart PedE (PP_2674), including linear and aromatic primary and secondary alcohols, as well as aldehydes, but only in the presence of lanthanide ions, including La³⁺, Ce³⁺, Pr³⁺, Sm³⁺, or Nd³⁺ Reporter assays revealed that PedH not only has a catalytic function but is also involved in the transcriptional regulation of <i>pedE</i> and <i>pedH</i>, most likely acting as a sensory module. Notably, the underlying regulatory network is responsive to as little as 1 to 10 nM lanthanum, a concentration assumed to be of ecological relevance. The present study further demonstrates that the PQQ-dependent oxidation system is crucial for efficient growth with a variety of volatile alcohols. From these results, we conclude that functional redundancy and inverse regulation of PedE and PedH represent an adaptive strategy of <i>P. putida</i> KT2440 to optimize growth with volatile alcohols in response to the availability of different lanthanides.<b>IMPORTANCE</b> Because of their low bioavailability, lanthanides have long been considered biologically inert. In recent years, however, the identification of lanthanides as a cofactor in methylotrophic bacteria has attracted tremendous interest among various biological fields. The present study reveals that one of the two PQQ-ADHs produced by the model organism <i>P. putida</i> KT2440 also utilizes lanthanides as a cofactor, thus expanding the scope of lanthanide-employing bacteria beyond the methylotrophs. Similar to the system described in methylotrophic bacteria, a complex regulatory network is involved in lanthanide-responsive switching between the two PQQ-ADHs encoded by <i>P. putida</i> KT2440. We further show that the functional production of at least one of the enzymes is crucial for efficient growth with several volatile alcohols. Overall, our study provides a novel understanding of the redundancy of PQQ-ADHs observed in many organisms and further highlights the importance of lanthanides for bacterial metabolism, particularly in soil environments. << Less

  MBio 8:e00570-e00570(2017) [PubMed] [EuropePMC]

  This publication is cited by 12 other entries.

• An inducible 1-butanol dehydrogenase, a quinohaemoprotein, is involved in the oxidation of butane by Pseudomonas butanovora.

  Vangnai A.S., Arp D.J.

  Butane-grown "Pseudomonas butanovora" expressed two soluble alcohol dehydrogenases (ADHs), an NAD(+)-dependent secondary ADH and an NAD(+)-independent primary ADH. Two additional NAD(+)-dependent secondary ADHs could be detected when cells were grown on 2-butanol and lactate. The inducible NAD(+)- ... >> More

  Butane-grown "Pseudomonas butanovora" expressed two soluble alcohol dehydrogenases (ADHs), an NAD(+)-dependent secondary ADH and an NAD(+)-independent primary ADH. Two additional NAD(+)-dependent secondary ADHs could be detected when cells were grown on 2-butanol and lactate. The inducible NAD(+)-independent 1-butanol dehydrogenase (BDH) of butane-grown cells was primarily responsible for 1-butanol oxidation in the butane metabolism pathway. BDH was purified to near homogeneity and identified as a quinohaemoprotein, containing, per mol enzyme, 1.0 mol pyrroloquinoline quinone (PQQ) and 0.25 mol haem c as prosthetic groups. BDH was synthesized as a monomer of approximately 66 kDa. It has a broad substrate range, including primary alcohols, secondary alcohols, aldehydes, C(4) diols and aromatic alcohols. It exhibited the lowest K:(m) (7+/-1 microM) and highest k(cat)/K:(m) (72x10(4) M(-1) s(-1)) value towards 1-butanol. BDH exhibited ferricyanide-dependent ADH activity. Calcium ions (up to 10 mM) increased BDH activity substantially. Two BDH internal amino acid sequences showed 73 and 62% identity and 83 and 66% similarity, respectively, when compared with an amino acid sequence of ethanol dehydrogenase from Comamonas testosteroni. The presence of the inducible BDH and secondary ADH may indicate that the terminal and subterminal oxidation pathways are involved in butane degradation of butane-grown "P. butanovora". << Less

  Microbiology 147:745-756(2001) [PubMed] [EuropePMC]

• Roles for the two 1-butanol dehydrogenases of Pseudomonas butanovora in butane and 1-butanol metabolism.

  Vangnai A.S., Sayavedra-Soto L.A., Arp D.J.

  Pseudomonas butanovora grown on butane or 1-butanol expresses two 1-butanol dehydrogenases, a quinoprotein (BOH) and a quinohemoprotein (BDH). BOH exhibited high affinity towards 1-butanol (K(m) = 1.7 +/-0.2 microM). BOH also oxidized butyraldehyde and 2-butanol (K(m) = 369 +/-85 microM and K(m) = ... >> More

  Pseudomonas butanovora grown on butane or 1-butanol expresses two 1-butanol dehydrogenases, a quinoprotein (BOH) and a quinohemoprotein (BDH). BOH exhibited high affinity towards 1-butanol (K(m) = 1.7 +/-0.2 microM). BOH also oxidized butyraldehyde and 2-butanol (K(m) = 369 +/-85 microM and K(m) = 662 +/-98 microM, respectively). The mRNA induction profiles of BOH and BDH at three different levels of 1-butanol, a nontoxic level (0.1 mM), a growth-supporting level (2 mM), and a toxic level (40 mM), were similar. When cells were grown in citrate-containing medium in the presence of different levels of 1-butanol, wild-type P. butanovora could tolerate higher levels of 1-butanol than the P. butanovora boh::tet strain and the P. butanovora bdh::kan strain. A model is proposed in which the electrons from 1-butanol oxidation follow a branched electron transport chain. BOH may be coupled to ubiquinone, with the electrons being transported to a cyanide-sensitive terminal oxidase. In contrast, electrons from BDH may be transferred to a terminal oxidase that is less sensitive to cyanide. The former pathway may function primarily in energy generation, while the latter may be more important in the detoxification of 1-butanol. << Less

  J. Bacteriol. 184:4343-4350(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Two distinct alcohol dehydrogenases participate in butane metabolism by Pseudomonas butanovora.

  Vangnai A.S., Arp D.J., Sayavedra-Soto L.A.

  The involvement of two primary alcohol dehydrogenases, BDH and BOH, in butane utilization in Pseudomonas butanovora (ATCC 43655) was demonstrated. The genes coding for BOH and BDH were isolated and characterized. The deduced amino acid sequence of BOH suggests a 67-kDa alcohol dehydrogenase contai ... >> More

  The involvement of two primary alcohol dehydrogenases, BDH and BOH, in butane utilization in Pseudomonas butanovora (ATCC 43655) was demonstrated. The genes coding for BOH and BDH were isolated and characterized. The deduced amino acid sequence of BOH suggests a 67-kDa alcohol dehydrogenase containing pyrroloquinoline quinone (PQQ) as cofactor and in the periplasm (29-residue leader sequence). The deduced amino acid sequence of BDH is consistent with a 70.9-kDa, soluble, periplasmic (37-residue leader sequence) alcohol dehydrogenase containing PQQ and heme c as cofactors. BOH and BDH mRNAs were induced whenever the cell's 1-butanol oxidation activity was induced. When induced with butane, the gene for BOH was expressed earlier than the gene for BDH. Insertional disruption of bdh or boh affected adversely, but did not eliminate, butane utilization by P. butanovora. The P. butanovora mutant with both genes boh and bdh inactivated was unable to grow on butane or 1-butanol. These cells, when grown in citrate and incubated in butane, developed butane oxidation capability and accumulated 1-butanol. The enzyme activity of BOH was characterized in cell extracts of the P. butanovora strain with bdh disrupted. Unlike BDH, BOH oxidized 2-butanol. The results support the involvement of two distinct NAD(+)-independent, PQQ-containing alcohol dehydrogenases, BOH (a quinoprotein) and BDH (a quinohemoprotein), in the butane oxidation pathway of P. butanovora. << Less

  J. Bacteriol. 184:1916-1924(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.