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Pyrroloquinoline Quinone Aza-Crown Ether Complexes as Biomimetics for Lanthanide and Calcium Dependent Alcohol Dehydrogenases*.

ABSTRACT: Understanding the role of metal ions in biology can lead to the development of new catalysts for several industrially important transformations. Lanthanides are the most recent group of metal ions that have been shown to be important in biology, that is, in quinone-dependent methanol dehydrogenases (MDH). Here we evaluate a literature-known pyrroloquinoline quinone (PQQ) and 1-aza-15-crown-5 based ligand platform as scaffold for Ca²⁺ , Ba²⁺ , La³⁺ and Lu³⁺ biomimetics of MDH and we evaluate the importance of ligand design, charge, size, counterions and base for the alcohol oxidation reaction using NMR spectroscopy. In addition, we report a new straightforward synthetic route (3 steps instead of 11 and 33 % instead of 0.6 % yield) for biomimetic ligands based on PQQ. We show that when studying biomimetics for MDH, larger metal ions and those with lower charge in this case promote the dehydrogenation reaction more effectively and that this is likely an effect of the ligand design which must be considered when studying biomimetics. To gain more information on the structures and impact of counterions of the complexes, we performed collision induced dissociation (CID) experiments and observe that the nitrates are more tightly bound than the triflates. To resolve the structure of the complexes in the gas phase we combined DFT-calculations and ion mobility measurements (IMS). Furthermore, we characterized the obtained complexes and reaction mixtures using Electron Paramagnetic Resonance (EPR) spectroscopy and show the presence of a small amount of quinone-based radical.

SUBMITTER: Vetsova VA

PROVIDER: S-EPMC8361747 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 Pseudomonas putida 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 pedE and pedH, 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 P. putida KT2440 to optimize growth with volatile alcohols in response to the availability of different lanthanides.IMPORTANCE 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 P. putida 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 P. putida 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.

Project description:Lanthanides are utilized by microbial methanol dehydrogenases, and it has been proposed that lanthanides may be important for other type I alcohol dehydrogenases. A triple mutant strain (mxaF xoxF1 xoxF2; named MDH-3), deficient in the three known methanol dehydrogenases of the model methylotroph Methylobacterium extorquens AM1, is able to grow poorly with methanol if exogenous lanthanides are added to the growth medium. When the gene encoding a putative quinoprotein ethanol dehydrogenase, exaF, was mutated in the MDH-3 background, the quadruple mutant strain could no longer grow on methanol in minimal medium with added lanthanum (La3+). ExaF was purified from cells grown with both calcium (Ca2+) and La3+ and with Ca2+ only, and the protein species were studied biochemically. Purified ExaF is a 126-kDa homodimer that preferentially binds La3+ over Ca2+ in the active site. UV-visible spectroscopy indicates the presence of pyrroloquinoline quinone (PQQ) as a cofactor. ExaF purified from the Ca2+-plus-La3+ condition readily oxidizes ethanol and has secondary activities with formaldehyde, acetaldehyde, and methanol, whereas ExaF purified from the Ca2+-only condition has minimal activity with ethanol as the substrate and activity with methanol is not detectable. The exaF mutant is not affected for growth with ethanol; however, kinetic and in vivo data show that ExaF contributes to ethanol metabolism when La3+ is present, expanding the role of lanthanides to multicarbon metabolism.ImportanceExaF is the most efficient PQQ-dependent ethanol dehydrogenase reported to date and, to our knowledge, the first non-XoxF-type alcohol oxidation system reported to use lanthanides as a cofactor, expanding the importance of lanthanides in biochemistry and bacterial metabolism beyond methanol dehydrogenases to multicarbon metabolism. These results support an earlier proposal that an aspartate residue near the catalytic aspartate residue may be an indicator of rare-earth element utilization by type I alcohol dehydrogenases.

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Pyrroloquinoline Quinone Aza-Crown Ether Complexes as Biomimetics for Lanthanide and Calcium Dependent Alcohol Dehydrogenases*.

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