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Pyruvate:quinone oxidoreductase (PQO) is a flavin‐containing peripheral membrane enzyme catalyzing the decarboxylation of pyruvate to acetate and CO2 with quinone as an electron acceptor. Here, we investigate PQO activity in Corynebacterium glutamicum, examine purified PQO, and describe the crystal structure of the native enzyme and a truncated version. The specific PQO activity was highest in stationary phase cells grown in complex medium, lower in cells grown in complex medium containing glucose or acetate, and lowest in cells grown in minimal acetate‐medium. A similar pattern with about 30‐fold higher specific PQO activities was observed in C. glutamicum with plasmid‐bound pqo expression under the control of the tac promoter, indicating that the differences in PQO activity are likely due to post‐transcriptional control. Continuous cultivation of C. glutamicum at dilution rates between 0.05 and 0.4 h−1 revealed a negative correlation between PQO activity and growth rate. Kinetic analysis of PQO enzymes purified from cells grown in complex or in minimal acetate‐medium revealed substantial differences in specific activity (72.3 vs. 11.9 U·mg protein−1) and turnover number (kcat: 440 vs. 78 s−1, respectively), suggesting post‐translational modifications affecting PQO activity. Structural analysis of PQO revealed a homotetrameric arrangement very similar to the Escherichia coli pyruvate oxidase PoxB except for the C‐terminal membrane binding domain, which exhibited a conformation markedly different from its PoxB counterpart. A truncated PQO variant lacking 17 C‐terminal amino acids showed higher affinity to pyruvate and was independent of detergent activation, highlighting the importance of the C‐terminus for enzyme activation and lipid binding. [ABSTRACT FROM AUTHOR]

The chemosensory pathway HtChe2 from the marine bacterium Halomonas titanicae KHS3 controls the activity of a diguanylate cyclase. Constitutive activation of this pathway results in colony morphology alterations and an increased ability to form biofilm. Such characteristics resemble the behavior of the Wsp pathway of Pseudomonas. In this work, we investigate the specificity of Htc10, the only chemoreceptor coded within the HtChe2 gene cluster. The purine derivatives guanine and hypoxanthine were identified as ligands of the recombinantly produced Htc10 ligand‐binding domain, with dissociation constants in the micromolar range, and its structure was solved by X‐ray protein crystallography. The sensor domain of Htc10 adopts a double Cache folding, with ligands bound to the membrane‐distal pocket. A high‐resolution structure of the occupied guanine‐binding pocket allowed the identification of residues involved in ligand recognition. Such residues were validated by site‐directed mutagenesis and isothermal titration calorimetry analyses of the protein variants. Moreover, heterologous expression of Htc10 in a Pseudomonas putida mutant lacking the native Wsp chemoreceptor promoted biofilm formation, a phenotype that was further enhanced by Htc10‐specific ligands. To our knowledge, this is the first description of binding specificity of a chemoreceptor that controls the activity of an associated diguanylate cyclase, opening the way for dynamic studies of the signaling behavior of this kind of sensory complex. [ABSTRACT FROM AUTHOR]