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Brassinosteroid insensitive1 (BRI1), a leucine-rich repeat receptor kinase, is responsible for the perception of the brassinosteroid (BR) phytohormone in plants. While recent evidence has implicated a naturally occurring Hordeum vulgareV. (barley) HvBRI1 kinase domain (KD) variant (H857R; “uzu” variation) in increased fungal disease resistance, the impact of the variation on receptor function and thus the mechanism by which disease resistance might be imparted remain enigmatic. Here, the functional implications of the uzu variation as well as the effects of newly identified naturally occurring Triticum aestivumL. (wheat) TaBRI1-KD variants are investigated. Recombinantly produced KDs of wild-type (WT) and uzu HvBRI1 were assessed for phosphorylation activity in vitro, yielding WT KMand VMAXvalues similar to those of other reports, but the uzu variation delayed saturation and reduced turnover levels. In silicomodeling of the H857R variation showed it to be surface-exposed and distal from the catalytic site. Further evaluation of three naturally occurring wheat TaBRI1 variants, A907T, A970V, and G1019R (barley numbering) identified in the A, B, and D subgenomic genes, respectively, highlighted a significant loss of activity for A907T. A907T is located on the same surface as the H857R variation and a negative regulatory phosphorylation site (T982) in Arabidopsis thalianaBRI1. A fourth variation, T1031A (barley numbering), unique to both subgenomic A proteins and localized to the BKI1 binding site, also decreased activity. The outcomes are discussed with respect to the predicted structural contexts of the variations and their implications with respect to mechanisms of action.

Tuberculosis remains one of the top causes of death worldwide, and combating its spread has been severely complicated by the emergence of drug-resistance mutations, highlighting the need for more effective drugs. Despite the resistance to isoniazid (INH) arising from mutations in the katGgene encoding the catalase-peroxidase KatG, most notably the S315T mutation, this compound is still one of the most powerful first-line antitubercular drugs, suggesting further pursuit of the development of tailored INH derivatives. The N′-acylated INH derivative with a long alkyl chain (INH-C10) has been shown to be more effective than INH against the S315T variant of Mycobacterium tuberculosis, but the molecular details of this activity enhancement are still unknown. In this work, we show that INH N′-acylation significantly reduces the rate of production of both isonicotinoyl radical and isonicotinyl–NAD by wild type KatG, but not by the S315T variant of KatG mirroring the in vivoeffectiveness of the compound. Restrained and unrestrained MD simulations of INH and its derivatives at the water/membrane interface were performed and showed a higher preference of INH-C10for the lipidic phase combined with a significantly higher membrane permeability rate (27.9 cm s–1), compared with INH-C2or INH (3.8 and 1.3 cm s–1, respectively). Thus, we propose that INH-C10is able to exhibit better minimum inhibitory concentration (MIC) values against certain variants because of its better ability to permeate through the lipid membrane, enhancing its availability inside the cell. MIC values of INH and INH-C10against two additional KatG mutations (S315N and D735A) revealed that some KatG variants are able to process INH faster than INH-C10into an effective antitubercular form (wtand S315N), while others show similar reaction rates (S315T and D735A). Altogether, our results highlight the potential of increased INH lipophilicity for treating INH-resistant strains.

The unusual Met-Tyr-Trp adduct composed of cross-linked side chains along with an associated mobile Arg is essential for catalase activity in catalase-peroxidases. In addition, acidic residues in the entrance channel, in particular an Asp and a Glu ∼7 and ∼15 Å, respectively, from the heme, significantly enhance catalase activity. The mechanism by which these channel carboxylates influence catalase activity is the focus of this work. Seventeen new variants with fewer and additional acidic residues have been constructed and characterized structurally and for enzymatic activity, revealing that their effect on activity is roughly inversely proportional to their distance from the heme and adduct, suggesting that the electrostatic potential of the heme cavity may be affected. A discrete group of protonable residues are contained within a 15 Å sphere surrounding the heme iron, and a computational analysis reveals that the pKaof the distal His112, alone, is modulated within the pH range of catalase activity by the remote acidic residues in a pattern consistent with its protonated form having a key role in the catalase reaction cycle. The electrostatic potential also impacts the catalatic reaction through its influence on the charged status of the Met-Tyr-Trp adduct.