H2S biogenesis by cystathionine beta-synthase: mechanism of inhibition by aminooxyacetic acid and unexpected role of serine.

Cystathionine beta-synthase (CBS) is a pivotal enzyme of the transsulfuration pathway responsible for diverting homocysteine to the biosynthesis of cysteine and production of hydrogen sulfide (H2S). Aberrant upregulation of CBS and overproduction of H2S contribute to pathophysiology of several diseases including cancer and Down syndrome. Therefore, pharmacological CBS inhibition has emerged as a prospective therapeutic approach. Here, we characterized binding and inhibitory mechanism of aminooxyacetic acid (AOAA), the most commonly used CBS inhibitor. We found that AOAA binds CBS tighter than its respective substrates and forms a dead-end PLP-bound intermediate featuring an oxime bond. Surprisingly, serine, but not cysteine, replaced AOAA from CBS and formed an aminoacrylate reaction intermediate, which allowed for the continuation of the catalytic cycle. Indeed, serine rescued and essentially normalized the enzymatic activity of AOAA-inhibited CBS. Cellular studies confirmed that AOAA decreased H2S production and bioenergetics, while additional serine rescued CBS activity, H2S production and mitochondrial function. The crystal structure of AOAA-bound human CBS showed a lack of hydrogen bonding with residues G305 and Y308, found in the serine-bound model. Thus, AOAA-inhibited CBS could be reactivated by serine. This difference may be important in a cellular environment in multiple pathophysiological conditions and may modulate the CBS-inhibitory activity of AOAA. In addition, our results demonstrate additional complexities of using AOAA as a CBS-specific inhibitor of H2S biogenesis and point to the urgent need to develop a potent, selective and specific pharmacological CBS inhibitor.

Phylogenetic Analysis with Prediction of Cofactor or Ligand Binding for Pseudomonas aeruginosa PAS and Cache Domains.

PAS domains are omnipresent building blocks of multidomain proteins in all domains of life. Bacteria possess a variety of PAS domains in intracellular proteins and the related Cache domains in periplasmic or extracellular proteins. PAS and Cache domains are predominant in sensory systems, often carry cofactors or bind ligands, and serve as dimerization domains in protein association. To aid our understanding of the wide distribution of these domains, we analyzed the proteome of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 in silico. The ability of this bacterium to survive under different environmental conditions, to switch between planktonic and sessile/biofilm lifestyle, or to evade stresses, notably involves c-di-GMP regulatory proteins or depends on sensory pathways involving multidomain proteins that possess PAS or Cache domains. Maximum likelihood phylogeny was used to group PAS and Cache domains on the basis of amino acid sequence. Conservation of cofactor- or ligand-coordinating amino acids aided by structure-based comparison was used to inform function. The resulting classification presented here includes PAS domains that are candidate binders of carboxylic acids, amino acids, fatty acids, flavin adenine dinucleotide (FAD), 4-hydroxycinnamic acid, and heme. These predictions are put in context to previously described phenotypic data, often generated from deletion mutants. The analysis predicts novel functions for sensory proteins and sheds light on functional diversification in a large set of proteins with similar architecture. IMPORTANCE To adjust to a variety of life conditions, bacteria typically use multidomain proteins, where the modular structure allows functional differentiation. Proteins responding to environmental cues and regulating physiological responses are found in chemotaxis pathways that respond to a wide range of stimuli to affect movement. Environmental cues also regulate intracellular levels of cyclic-di-GMP, a universal bacterial secondary messenger that is a key determinant of bacterial lifestyle and virulence. We study Pseudomonas aeruginosa, an organism known to colonize a broad range of environments that can switch lifestyle between the sessile biofilm and the planktonic swimming form. We have investigated the PAS and Cache domains, of which we identified 101 in 70 Pseudomonas aeruginosa PAO1 proteins, and have grouped these by phylogeny with domains of known structure. The resulting data set integrates sequence analysis and structure prediction to infer ligand or cofactor binding. With this data set, functional predictions for PAS and Cache domain-containing proteins are made.

Differential impact on motility and biofilm dispersal of closely related phosphodiesterases in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the transition between planktonic and biofilm lifestyles is modulated by the intracellular secondary messenger cyclic dimeric-GMP (c-di-GMP) in response to environmental conditions. Here, we used gene deletions to investigate how the environmental stimulus nitric oxide (NO) is linked to biofilm dispersal, focusing on biofilm dispersal phenotype from proteins containing putative c-di-GMP turnover and Per-Arnt-Sim (PAS) sensory domains. We document opposed physiological roles for the genes ΔrbdA and Δpa2072 that encode proteins with identical domain structure: while ΔrbdA showed elevated c-di-GMP levels, restricted motility and promoted biofilm formation, c-di-GMP levels were decreased in Δpa2072, and biofilm formation was inhibited, compared to wild type. A second pair of genes, ΔfimX and ΔdipA, were selected on the basis of predicted impaired c-di-GMP turnover function: ΔfimX showed increased, ΔdipA decreased NO induced biofilm dispersal, and the genes effected different types of motility, with reduced twitching for ΔfimX and reduced swimming for ΔdipA. For all four deletion mutants we find that NO-induced biomass reduction correlates with increased NO-driven swarming, underlining a significant role for this motility in biofilm dispersal. Hence P. aeruginosa is able to differentiate c-di-GMP output using structurally highly related proteins that can contain degenerate c-di-GMP turnover domains.

An embedded lipid in the multidrug transporter LmrP suggests a mechanism for polyspecificity.

Multidrug efflux pumps present a challenge to the treatment of bacterial infections, making it vitally important to understand their mechanism of action. Here, we investigate the nature of substrate binding within Lactococcus lactis LmrP, a prototypical multidrug transporter of the major facilitator superfamily. We determined the crystal structure of LmrP in a ligand-bound outward-open state and observed an embedded lipid in the binding cavity of LmrP, an observation supported by native mass spectrometry analyses. Molecular dynamics simulations suggest that the anionic lipid stabilizes the observed ligand-bound structure. Mutants engineered to disrupt binding of the embedded lipid display reduced transport of some, but not all, antibiotic substrates. Our results suggest that a lipid within the binding cavity could provide a malleable hydrophobic component that allows adaptation to the presence of different substrates, helping to explain the broad specificity of this protein and possibly other multidrug transporters.

Dimerisation induced formation of the active site and the identification of three metal sites in EAL-phosphodiesterases.

The bacterial second messenger cyclic di-3',5'-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825EAL) and PA1727 (MucREAL). Different dimerisation interfaces allow us to identify interface independent principles of enzyme regulation. Unlike previously characterised two-metal binding EAL-phosphodiesterases, PA3825EAL in complex with pGpG provides a model for a third metal site. The third metal is positioned to stabilise the negative charge of the 5'-phosphate, and thus three metals could be required for catalysis in analogy to other nucleases. This newly uncovered variation in metal coordination may provide a further level of bacterial PDE regulation.