Structural characterization of a hypothetical protein: a potential agent involved in trimethylamine metabolism in Catenulispora acidiphila

Catenulispora acidiphila is a newly identified lineage of actinomycetes that produces antimicrobial activities and represents a promising source of novel antibiotics and secondary metabolites. Among the discovered protein coding genes, 68 % were assigned a putative function, while the remaining 32 % are genes encoding “hypothetical” proteins. Caci_0382 is one of the “hypothetical” proteins that has very few homologs. Sequence analysis shows that the protein belongs to the NTF2-like protein family. The structure of Caci_0382 demonstrates that it shares the same fold and has a similar active site as limonene-1,2-epoxide hydrolase, which suggests that it may have a related function. Using a fluorescence thermal shift assay, we identified stabilizing compounds that suggest potential natural ligands of Caci_0382. Using this information, we determined the crystal structure in complex with trimethylamine to provide a better understanding of the function of this uncharacterized protein. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10969-014-9176-z) contains supplementary material, which is available to authorized users

Structural and biochemical studies of novel Aldo-keto Reductases (AKRs) for the biocatalytic conversion of 3-hydroxybutanal to 1,3-butanediol

The non-natural alcohol 1,3-butanediol (1,3-BDO) is a valuable building block for the synthesis of various polymers. One of the potential pathways for the biosynthesis of 1,3-BDO includes the biotransformation of acetaldehyde to 1,3-BDO via 3-hydroxybutanal (3-HB) using aldolases and aldo-keto reductases. This pathway requires an aldo-keto reductase (AKR) selective for 3-HB, but inactive toward acetaldehyde, so it can be used for one pot synthesis. In this work, we screened over 20 purified uncharacterized AKRs for 3-HB reduction and identified 10 enzymes with significant activity and nine proteins with detectable activity. PA1127 from Pseudomonas aeruginosa showed the highest activity and was selected for comparative studies with STM2406 from Salmonella typhimurium, for which we have determined the crystal structure. Both AKRs used NADPH as cofactor, reduced a broad range of aldehydes, and showed low activity toward acetaldehyde. The crystal structures of STM2406 in complex with cacodylate or NADPH revealed the active site with bound molecules of a substrate mimic or cofactor. Site-directed mutagenesis of STM2406 and PA1127 identified the key residues important for activity against 3-HB and aromatic aldehydes, which include the residues of the substrate binding pocket and C-terminal loop. Our results revealed that the replacement of the STM2406 Asn65 by Met enhanced both activity and affinity of this protein toward 3-HB resulting in a seven-fold increase in kcat/Km. Our work provided further insights into the molecular mechanisms of substrate selectivity of AKRs and rational design of these enzymes towards new substrates. Importance In this study, we identified several aldo-keto reductases with significant activity in the reduction of 3-hydroxybutanal to 1,3-BDO, an important commodity chemical. Biochemical and structural studies of these enzymes revealed the key catalytic and substrate binding residues including the two structural determinants necessary for high activity in the biosynthesis of 1,3-BDO. This work expands our understanding of the molecular mechanisms of substrate selectivity of AKRs and the potential for protein engineering of these enzymes for applications in the biocatalytic production of 1,3-BDO and other valuable chemicals

Development of Novel In Vivo Chemical Probes to Address CNS Protein Kinase Involvement in Synaptic Dysfunction

Serine-threonine protein kinases are critical to CNS function, yet there is a dearth of highly selective, CNS-active kinase inhibitors for in vivo investigations. Further, prevailing assumptions raise concerns about whether single kinase inhibitors can show in vivo efficacy for CNS pathologies, and debates over viable approaches to the development of safe and efficacious kinase inhibitors are unsettled. It is critical, therefore, that these scientific challenges be addressed in order to test hypotheses about protein kinases in neuropathology progression and the potential for in vivo modulation of their catalytic activity. Identification of molecular targets whose in vivo modulation can attenuate synaptic dysfunction would provide a foundation for future disease-modifying therapeutic development as well as insight into cellular mechanisms. Clinical and preclinical studies suggest a critical link between synaptic dysfunction in neurodegenerative disorders and the activation of p38αMAPK mediated signaling cascades. Activation in both neurons and glia also offers the unusual potential to generate enhanced responses through targeting a single kinase in two distinct cell types involved in pathology progression. However, target validation has been limited by lack of highly selective inhibitors amenable to in vivo use in the CNS. Therefore, we employed high-resolution co-crystallography and pharmacoinformatics to design and develop a novel synthetic, active site targeted, CNS-active, p38αMAPK inhibitor (MW108). Selectivity was demonstrated by large-scale kinome screens, functional GPCR agonist and antagonist analyses of off-target potential, and evaluation of cellular target engagement. In vitro and in vivo assays demonstrated that MW108 ameliorates beta-amyloid induced synaptic and cognitive dysfunction. A serendipitous discovery during co-crystallographic analyses revised prevailing models about active site targeting of inhibitors, providing insights that will facilitate future kinase inhibitor design. Overall, our studies deliver highly selective in vivo probes appropriate for CNS investigations and demonstrate that modulation of p38αMAPK activity can attenuate synaptic dysfunction

Protein target highlights in CASP15: Analysis of models by structure providers

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology

Structural Basis for c-di-GMP-Mediated Inside-Out Signaling Controlling Periplasmic Proteolysis

X-ray crystallographic structural analyses of the bacterial transmembrane receptor LapD identify conserved molecular mechanisms that control biofilm formation in response to changes in the intracellular levels of the second messenger c-di-GMP