Rajesh Nagarajan

Evolution of Acyl-Substrate Recognition by a Family of Acyl-Homoserine Lactone Synthases

Members of the LuxI protein family catalyze synthesis of acyl-homoserine lactone (acyl-HSL) quorum sensing signals from S-adenosyl-L-methionine and an acyl thioester. Some LuxI family members prefer acyl-CoA, and others prefer acyl-acyl carrier protein (ACP) as the acyl-thioester substrate. We sought to understand the evolutionary history and mechanisms mediating this substrate preference. Our phylogenetic and motif analysis of the LuxI acyl-HSL synthase family indicates that the acyl-CoA-utilizing enzymes evolved from an acyl-ACP-utilizing ancestor. To further understand how acyl-ACPs and acyl-CoAs are recognized by acyl-HSL synthases we studied BmaI1, an octanoyl-ACP-dependent LuxI family member from Burkholderia mallei, and BjaI, an isovaleryl-CoA-dependent LuxI family member from Bradyrhizobium japonicum. We synthesized thioether analogs of their thioester acyl-substrates to probe recognition of the acyl-phosphopantetheine moiety common to both acyl-ACP and acyl-CoA substrates. The kinetics of catalysis and inhibition of these enzymes indicate that they recognize the acyl-phosphopantetheine moiety and they recognize non-preferred substrates with this moiety. We find that CoA substrate utilization arose through exaptation of acyl-phosphopantetheine recognition in this enzyme family.

Am I a STEM professional? Documenting STEM student professional identity development

Post-secondary education is expected to substantially contribute to the cognitive growth and professional achievement of students studying science, technology, engineering, and mathematics (STEM). Yet, there is limited understanding of how students studying STEM develop a professional identity. We used the lens of self-authorship to develop a model for STEM student professional identity development. We applied the model to frame our assessment of the relationship between the level of STEM students’ perceptions of their professional identities and their educational experiences, learning preferences, and comfort with faculty interactions. We found a misalignment between students’ perception of themselves as professionals and the expectations for their actions in professional situations. We also found that students engaged in learning activities similar to the activities of STEM professionals communicated higher levels of professional identity development. We provide implications for our research and directions for ongoing investigations.

Acyl-ACP Substrate Recognition in Burkholderia mallei BmaI1 Acyl-Homoserine Lactone Synthase

The acyl-homoserine lactone (AHL) autoinducer mediated quorum sensing regulates virulence in several pathogenic bacteria. The hallmark of an efficient quorum sensing system relies on the tight specificity in the signal generated by each bacterium. Since AHL signal specificity is derived from the acyl-chain of the acyl-ACP (ACP = acyl carrier protein) substrate, AHL synthase enzymes must recognize and react with the native acyl-ACP with high catalytic efficiency while keeping reaction rates with non-native acyl-ACPs low. The mechanism of acyl-ACP substrate recognition in these enzymes, however, remains elusive. In this study, we investigated differences in catalytic efficiencies for shorter and longer chain acyl-ACP substrates reacting with an octanoyl-homoserine lactone synthase Burkholderia mallei BmaI1. With the exception of two-carbon shorter hexanoyl-ACP, the catalytic efficiencies of butyryl-ACP, decanoyl-ACP, and octanoyl-CoA reacting with BmaI1 decreased by greater than 20-fold compared to the native octanoyl-ACP substrate. Furthermore, we also noticed kinetic cooperativity when BmaI1 reacted with non-native acyl-donor substrates. Our kinetic data suggest that non-native acyl-ACP substrates are unable to form a stable and productive BmaI1·acyl-ACP·SAM ternary complex and are thus effectively discriminated by the enzyme. These results offer insights into the molecular basis of substrate recognition for the BmaI1 enzyme.

Molecular Basis for the Substrate Specificity of Quorum Signal Synthases

Significance These first structures of a homoserine-lactone quorum-signal synthase bound to various substrates and analogs help to provide a molecular rationale for understanding acyl chain specificity. Based on the structural data, we show how different clades of signal synthases can accommodate their cognate acyl–CoA ligands. Lastly, the elucidation of the reaction mechanism for the signal synthase may provide a rationale for the design of therapeutic small-molecule antagonists. In several Proteobacteria, LuxI-type enzymes catalyze the biosynthesis of acyl–homoserine lactones (AHL) signals using S-adenosyl–l-methionine and either cellular acyl carrier protein (ACP)-coupled fatty acids or CoA–aryl/acyl moieties as progenitors. Little is known about the molecular mechanism of signal biosynthesis, the basis for substrate specificity, or the rationale for donor specificity for any LuxI member. Here, we present several cocrystal structures of BjaI, a CoA-dependent LuxI homolog that represent views of enzyme complexes that exist along the reaction coordinate of signal synthesis. Complementary biophysical, structure–function, and kinetic analysis define the features that facilitate the unusual acyl conjugation with S-adenosylmethionine (SAM). We also identify the determinant that establishes specificity for the acyl donor and identify residues that are critical for acyl/aryl specificity. These results highlight how a prevalent scaffold has evolved to catalyze quorum signal synthesis and provide a framework for the design of small-molecule antagonists of quorum signaling.

A Comparative Analysis of Acyl-Homoserine Lactone Synthase Assays.

Quorum sensing is cell‐to‐cell communication that allows bacteria to coordinate attacks on their hosts by inducing virulent gene expression, biofilm production, and other cellular functions, including antibiotic resistance. AHL synthase enzymes synthesize N‐acyl‐l‐homoserine lactones, commonly referred to as autoinducers, to facilitate quorum sensing in Gram‐negative bacteria. Studying the synthases, however, has proven to be a difficult road. Two assays, including a radiolabeled assay and a colorimetric (DCPIP) assay are well‐documented in literature to study AHL synthases. In this paper, we describe additional methods that include an HPLC‐based, C−S bond cleavage and coupled assays to investigate this class of enzymes. In addition, we compare and contrast each assay for both acyl‐CoA‐ and acyl‐ACP‐utilizing synthases. The expanded toolkit described in this study should facilitate mechanistic studies on quorum sensing signal synthases and expedite discovery of antivirulent compounds.

Enzymatic Assays to Investigate Acyl-Homoserine Lactone Autoinducer Synthases

Bacteria use chemical molecules called autoinducers as votes to poll their numerical strength in a colony. This polling mechanism, commonly referred to as quorum sensing, enables bacteria to build a social network and provide a collective response for fighting off common threats. In Gram-negative bacteria, AHL synthases synthesize acyl-homoserine lactone (AHL) autoinducers to turn on the expression of several virulent genes including biofilm formation, protease secretion, and toxin production. Therefore, inhibiting AHL signal synthase would limit quorum sensing and virulence. In this chapter, we describe four enzymatic methods that could be adopted to investigate a broad array of AHL synthases. The enzymatic assays described here should accelerate our mechanistic understanding of quorum-sensing signal synthesis that could pave the way for discovery of potent antivirulence compounds.

Structure–Function Analyses of the N-Butanoyl l-Homoserine Lactone Quorum-Sensing Signal Define Features Critical to Activity in RhlR

Pseudomonas aeruginosa is an opportunistic pathogen that coordinates the production of many virulence phenotypes at high population density via quorum sensing (QS). The LuxR-type receptor RhlR plays an important role in the P. aeruginosa QS process, and there is considerable interest in the development of chemical approaches to modulate the activity of this protein. RhlR is activated by a simple, low molecular weight N-acyl l-homoserine lactone signal, N-butanoyll-homoserine lactone (BHL). Despite the emerging prominence of RhlR in QS pathways, there has been limited exploration of the chemical features of the BHL scaffold that are critical to its function. In the current study, we sought to systematically delineate the structure-activity relationships (SARs) driving BHL activity for the first time. A focused library of BHL analogues was designed, synthesized, and evaluated in cell-based reporter gene assays for RhlR agonism and antagonism. These investigations allowed us to define a series of SARs for BHL-type ligands and identify structural motifs critical for both activation and inhibition of the RhlR receptor. Notably, we identified agonists that have ∼10-fold higher potencies in RhlR relative to BHL, are highly selective for RhlR agonism over LasR, and are active in the P. aeruginosa background. These compounds and the SARs reported herein should pave a route toward new chemical strategies to study RhlR in P. aeruginosa.