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

Abstract

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.

Biochemical and Structural Characterization of XoxG and XoxJ and Their Roles in Lanthanide‐Dependent Methanol Dehydrogenase Activity

“…XoxF was purified from its native levels as described, by ammonium sulfate precipitation, cation‐exchange chromatography, and size‐exclusion chromatography (SEC), from M. extorquens AM1 cultures grown in the presence of 1 μ m LaCl 3 , CeCl 3 , or NdCl 3 (Figure S1 in the Supporting Information). The UV/visible spectra of these proteins are shown in Figure S2, with the characteristic absorption band at ≈360 nm and a shoulder at ≈410 nm indicative of the metal‐bound PQQ cofactor . The protein yields, metal contents, and A 280 nm / A 359 nm ratios for each XoxF were similar (Table S1), thus indicating similar cofactor loading.…”

Laboratory evolution reveals the metabolic and regulatory basis of ethylene glycol metabolism byPseudomonas putidaKT2440

“…Initially, the diol is converted into glyoxylate in a series of oxidation reactions catalysed by a set of redundant dehydrogenases, with PP_0545, PedI, PedE and PedH as predominant enzymes (Fig. 1A) (Mückschel et al, 2012;Wehrmann et al, 2017). Further oxidation to oxalate also occurs in wholecell biotransformations, but generally only relatively small traces of this dead end-product are observed (Mückschel et al, 2012).…”

Isolation and genomic characterization ofNovimethylophilus kurashikiensisgen. nov. sp. nov., a new lanthanide‐dependent methylotrophic species ofMethylophilaceae

“…The ability of ExaF to use Ln 31 as a cofactor, PQQ-dependent ethanol dehydrogenase in Methylobacterium extorquens AM1, enlarged the importance of Ln 31 to multicarbon metabolism . The finding of PedH, Ln 31 -dependent PQQ-alcohol dehydrogenase in a nonmethylotrophic bacterium, Pseudomonas putida KT2440, expanded the range of Ln 31 utilizing bacteria beyond the methylotrophs (Wehrmann et al, 2017).…”