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The 3‐(3‐hydroxyalkanoyloxy)alkanoate (HAA) synthase RhlA is an essential enzyme involved in the biosynthesis of HAAs in Pseudomonas and Burkholderia species. RhlA modulates the aliphatic chain length in rhamnolipids, conferring distinct physicochemical properties to these biosurfactants exhibiting promising industrial and pharmaceutical value. A detailed molecular understanding of substrate specificity and catalytic performance in RhlA could offer protein engineering tools to develop designer variants involved in the synthesis of novel rhamnolipid mixtures for tailored eco‐friendly products. However, current directed evolution progress remains limited due to the absence of high‐throughput screening methodologies and lack of an experimentally resolved RhlA structure. In the present work, we used comparative modeling and chimeric‐based approaches to perform a comprehensive semi‐rational mutagenesis of RhlA from Pseudomonas aeruginosa. Our extensive RhlA mutational variants and chimeric hybrids between the Pseudomonas and Burkholderia homologs illustrate selective modulation of rhamnolipid alkyl chain length in both Pseudomonas aeruginosa and Burkholderia glumae. Our results also demonstrate the implication of a putative cap‐domain motif that covers the catalytic site of the enzyme and provides substrate specificity to RhlA. This semi‐rational mutant‐based survey reveals promising 'hot‐spots' for the modulation of RL congener patterns and potential control of enzyme activity, in addition to uncovering residue positions that modulate substrate selectivity between the Pseudomonas and Burkholderia functional homologs. Database: Model data are available in the PMDB database under the accession number PM0081867. [ABSTRACT FROM AUTHOR]

Key points The renin-angiotensin system plays a key role in cardiovascular physiology and its overactivation has been implicated in the pathogenesis of several major cardiovascular diseases., There is growing evidence that angiotensin II (Ang-II) may function as an intracellular peptide to activate intracellular/nuclear receptors and their downstream signalling effectors independently of cell surface receptors., Current methods used to study intracrine Ang-II signalling are limited to indirect approaches because of a lack of selective intracellularly-acting probes., Here, we present novel photoreleasable Ang-II analogues used to probe intracellular actions with spatial and temporal precision., The photorelease of intracellular Ang-II causes nuclear and cytosolic calcium mobilization and initiates the de novo synthesis of RNA in cardiac cells, demonstrating the application of the method., Abstract Several lines of evidence suggest that intracellular angiotensin II (Ang-II) contributes to the regulation of cardiac contractility, renal salt reabsorption, vascular tone and metabolism; however, work on intracrine Ang-II signalling has been limited to indirect approaches because of a lack of selective intracellularly-acting probes. Here, we aimed to synthesize and characterize cell-permeant Ang-II analogues that are inactive without uncaging, but release active Ang-II upon exposure to a flash of UV-light, and act as novel tools for use in the study of intracrine Ang-II physiology. We prepared three novel caged Ang-II analogues, [Tyr(DMNB)4]Ang-II, Ang-II-ODMNB and [Tyr(DMNB)4]Ang-II-ODMNB, based upon the incorporation of the photolabile moiety 4,5-dimethoxy-2-nitrobenzyl (DMNB). Compared to Ang-II, the caged Ang-II analogues showed 2-3 orders of magnitude reduced affinity toward both angiotensin type-1 (AT1R) and type-2 (AT2R) receptors in competition binding assays, and greatly-reduced potency in contraction assays of rat thoracic aorta. After receiving UV-irradiation, all three caged Ang-II analogues released Ang-II and potently induced the contraction of rat thoracic aorta. [Tyr(DMNB)4]Ang-II showed the most rapid photolysis upon UV-irradiation and was the focus of subsequent characterization. Whereas Ang-II and photolysed [Tyr(DMNB)4]Ang-II increased ERK1/2 phosphorylation (via AT1R) and cGMP production (AT2R), caged [Tyr(DMNB)4]Ang-II did not. Cellular uptake of [Tyr(DMNB)4]Ang-II was 4-fold greater than that of Ang-II and significantly greater than uptake driven by the positive-control HIV TAT(48-60) peptide. Intracellular photolysis of [Tyr(DMNB)4]Ang-II induced an increase in nucleoplasmic Ca2+ ([Ca2+]n), and initiated 18S rRNA and nuclear factor kappa B mRNA synthesis in adult cardiac cells. We conclude that caged Ang-II analogues represent powerful new tools for use in the selective study of intracrine signalling via Ang-II. [ABSTRACT FROM AUTHOR]

Studies the development of agonists of endothelin-1 (ET-1) exhibiting selectivity towards ET[sub A] receptors. Description of the first compounds showing a significant bioactivity in an ET[sub A] pharmacological preparation; Implications for pharmacological research.