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Bacteria secrete siderophores to sequester the scarce iron in the environments, then the iron is transported into the cell in a siderophore-complexed form, which can be released by siderophore-interacting protein (SIP). Vibrio species comprise an array of serious pathogens, whose iron releasing process by SIP remains poorly understood. Herein, we report the high-resolution (1.2 Å) structure of Vibrio anguillarum SIP (VaSIP) in complex with FAD, representing the first structure of Vibrio SIP. VaSIP consists of a FAD-bound β-barrel domain and a Rossmann-fold domain connected by a linker, like other subgroup I SIPs. FAD is bound to the inter-domain cavity by aromatic stacking and hydrogen bonding interactions. Structural comparison indicated a modified NAD(P)H-binding motif (DxTA–EVL–GE) for subgroup I SIPs. The putative siderophore-binding pocket of VaSIP contains three lysines to form the basic triad to bind siderophore. Phylogenetic analysis shows Vibrio SIPs are mainly divided into two clades, represented by VaSIP and Vibrio cholerae ViuB, respectively. Interestingly, the two clades adopt distinct siderophore-binding basic triads, suggesting functional divergence among Vibrio SIPs. Our results shed light on the structural and phylogenetic characteristics of Vibrio SIPs, providing molecular basis for understanding Vibrio iron metabolism and designing anti- Vibrio drugs. • The first structure of Vibrio siderophore-interacting protein (SIP) was reported. • The FAD-, NAD(P)H- and siderophore-binding regions are analyzed. • Vibrio SIPs are mainly divided into two clades, with the basic triads as markers. [ABSTRACT FROM AUTHOR]

Antibiotic resistance is becoming a global threat and overuse of antibiotics in aquaculture disease control worsens the situation. To reduce the risk of drug resistance developed in aquaculture, safer biocontrol programs are needed. Antivirulence therapy, with less chance for developing drug resistance, is a promising approach. To facilitate antivirulence inhibitor design against Vibrio anguillarum , a serious aquaculture pathogen, we present crystal structures for isochorismatase domains of AngB and VabB, which are required to synthesize siderophore, a critical virulence factor. Both structures are highly similar to known isochorismatases in fold and active site, therefore we conclude inhibitors for isochorismatases can be developed in a common framework. The structural information will improve design of virulence inhibitors against Vibrio anguillarum . We also firstly report that isochorismatase family could bind endogenous metabolite during the hetero-expression process, which is likely nicotinic acid, nicotinamide or pyrazinic acid, based on structural analysis and affinity prediction. Taken together, our results provide precise structural information of isochorismatase domains for antivirulence inhibitor design against Vibrio anguillarum . [ABSTRACT FROM AUTHOR]

l -serine ammonia-lyase, as a member of the β-family of pyridoxal-5′-phosphate (PLP) dependent enzymes, catalyzes the conversion of l -serine ( l -threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of l -serine ammonia-lyase from Rhizomucor miehei ( Rm SDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of Rm SDH had the characteristic β-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/β structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147 and Thr312) were discovered at the catalytic site of Rm SDH by comparison of structures of Rm SDH and other reported eukaryotic l -serine ammonia-lyases. Optimal pH and temperature of the purified Rm SDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0–9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal l -serine ammonia-lyase. It will be useful to study the catalytic mechanism of β-elimination enzymes and will provide a basis for further enzyme engineering. [ABSTRACT FROM AUTHOR]

To clarify the mechanism of the stimulatory effect of statins on bone formation, we investigated the effect of simvastatin, a widely used statin, on osteoblastic and adipocytic differentiation in primary cultured mouse bone marrow stromal cells (BMSCs). Simvastatin treatment enhanced the expression level of mRNA for osteocalcin and protein for osteocalcin and osteopontin, and increased alkaline phosphatase activity significantly (p<0.05). After BMSCs were exposed to an adipocyte differentiation agonist, Oil Red O staining, fluorescence activated cell sorting, and decreased expression level of lipoprotein lipase mRNA showed that treatment with simvastatin significantly inhibits adipocytic differentiation compared to controls that did not receive simvastatin (p<0.05). Lastly, we found that simvastatin induces high expression of BMP2 in BMSCs. These observations suggested that simvastatin acts on BMSCs to enhance osteoblastic differentiation and inhibits adipocytic differentiation; this effect is at least partially mediated by inducing BMP2 expression in BMSCs. [Copyright &y& Elsevier]

Abstract Extracellular proteases are often produced as pre-pro-enzyme and then undergo multiple processing steps to mature into the active form. The protease Epp, a virulent factor of Vibrio anguillarum, belongs to this family. Its maturation might be regulated by Ca2+ via its polycystic kidney disease (PKD) domain, but the molecular mechanism is unknown. Herein, we report the crystal structure of the first PKD domain from V. anguillarum Epp (Epp-PKD1) and its specific Ca2+-binding capacity. Epp-PKD1 exists as a monomer, consisting of seven β-strands which form two β-sheets stacking with each other. One Ca2+ is bound by the residues Asn3, Gln4, Asp27, Asp29, Asp68 and a water molecule with a pentagonal bipyramidal geometry. Incubating the apo Epp-PKD1 with Ca2+ but not Mg2+, Mn2+, or Zn2+, enhances the thermal and chemical stability of Epp-PKD1, indicating its specific binding to Ca2+. Epp-PKD1 shares high similarity in both sequence and overall structure with that of Vibrio cholerae PrtV, a homologous protease of Epp, however, they differ in the oligomeric state and local structure at the Ca2+-binding site, suggesting maturation of PrtV and Epp might be differently regulated by Ca2+. Likely, proteases may take advantage of the structural diversity in PKD domains to tune their Ca2+-regulated maturation process. Highlights • Crystal structure of the first PKD domain of Vibrio anguillarum Epp is determined. • Epp-PKD1 contains a pentagonal-bipyramidal Ca2+-binding site. • Specific binding to Ca2+ increases conformational stability of Epp-PKD1. • Epp-PKD1 differs in the oligomeric state and Ca2+ coordination from the homologous PrtV-PKD1. [ABSTRACT FROM AUTHOR]

RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39. [ABSTRACT FROM AUTHOR]