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Glucose isomerase (GI) is extensively used in the food industry for production of high-fructose corn syrup and for the production of biofuels and other renewable chemicals. Structure-based studies on GI inhibitors are important for improving its efficiency in industrial applications. Here, we report the subatomic crystal structure of Streptomyces rubiginosus GI (SruGI) complexed with its inhibitor, xylitol, at 0.99 Å resolution. Electron density map and temperature factor analysis showed partial binding of xylitol to the M1 metal binding site of SruGI, providing two different conformations of the metal binding site and the substrate binding channel. The xylitol molecule induced a conformational change in the M2 metal ion-interacting Asp255 residue, which subsequently led to a conformational change in the side chain of Asp181 residue. This led to the positional shift of Pro25 by 1.71 Å and side chain rotation of Phe26 by 21°, where located on the neighboring protomer in tetrameric SruGI. The conformation change of these two residues affect the size of the substrate-binding channel of GI. Therefore, xylitol binding to M1 site of SruGI induces not only a conformational changes of the metal-binding site, but also conformational change of substrate-binding channel of the tetrameric SruGI. These results expand our knowledge about the mechanism underlying the inhibitory effect of xylitol on GI. • The subatomic structure of S. rubiginosus glucose isomerase complexed with xylitol was determined. • Xylitol induces conformational changes in the M2 metal ion-interacting residues. • Xylitol induces conformational changes in the substrate binding channel. • Partial binding of xylitol to the M1 metal binding site of SruGI showed two different conformations. [ABSTRACT FROM AUTHOR]

The fluorescence of fluorescent proteins (FPs) is quenched when they are exposed to certain transition metals, which makes them promising receptor materials for metal biosensors. In this study, we report the spectroscopic analysis of metal-induced fluorescence quenching of the fluorescent protein ZsGreen from Zoanthus sp. The fluorescence of ZsGreen was reduced to 2%, 1%, and 20% of its original intensity by Fe2+, Fe3+, and Cu2+, respectively. Metal titration experiments indicated that the dissociation constants of Fe2+, Fe3+, and Cu2+ for ZsGreen were 11.5, 16.3, and 68.2 μM, respectively. The maximum binding capacities of ZsGreen for Fe2+, Fe3+, and Cu2+ were 103.3, 102.2, and 82.9, respectively. Reversibility experiments indicated that the fluorescence of ZsGreen, quenched by Fe2+ and Fe3+, could be recovered, but only to about 15% of its original intensity, even at a 50-fold molar excess of EDTA. In contrast, the fluorescence quenched by Cu2+ could be recovered up to 89.47% of its original intensity at a Cu2+: EDTA ratio of 1:5. The homology model of ZsGreen revealed that the protein does not share any metal-binding sites with previously reported FPs, suggesting that ZsGreen contains unprecedented binding sites for fluorescence quenching metal ions. [ABSTRACT FROM AUTHOR]

Abstract: The bifunctional glycoside hydrolase enzyme, CelM2, is able to hydrolyze glucan and xylan effectively. The crystal structure of this protein has been determined, providing useful sequential and structural information [K.H. Nam, S.J. Kim, K.Y. Hwang, Crystal structure of CelM2, a bifunctional glucanase–xylanase protein from a metagenome library, Biochem. Biophys. Res. Commun. 383 (2009) 183–186]. In addition, this protein is a good model for understanding bifunctional enzymes, and it will provide information relevant for genetic engineering that will be useful in the design of bifunctional proteins. However, previous structural characterization was not sufficient to develop an understanding of the metal ion and substrate-binding moiety. Herein, we determined the metal-binding site of CelM2 using zinc ions. Our results revealed that the zinc ions participate in the crystallographic packing and enzyme folding of the external region of the TIM-like barrel domain. Based on our structure, zinc ions induce the passive form of the CAP region at the catalytic cleft of the CelM2 protein. Moreover, glucose was bound to the CelM2 structure at the catalytic site. This structure provides the binding moiety that binds to the hydroxyl group of substrates such as cellulose. In addition, a structural comparison of celM2 with Cel44 provides a good model of the binding mode of CelM2. Thus, our study represents a novel structural characterization of the metal-binding site and the structure of the complex formed between CelM2 and its substrate. [Copyright &y& Elsevier]

Abstract: Hormone-sensitive lipase (HSL) plays an important role in the regulation of rodent fat cell lipolysis. It is regarded as an adipose tissue-specific enzyme whose sole metabolic role is the catalysis of hormone-stimulated lipolysis in mammalian cells. In this report we describe the functional and structural analysis of an EstE5 protein from a soil metagenome library. Function analysis results indicated that EstE5 preferentially hydrolyzes short-chain ester compounds, and our kinetic studies revealed the optimal pH and temperature. Based on the structural analysis, we defined the active site and the binding pocket. Structurally, EstE5 belongs to the HSL family and these structural studies may have applications in the production of value-added products, including pharmaceuticals. [Copyright &y& Elsevier]

Glucose isomerase (GI) is an important enzyme that is widely used in industrial applications, such as in the production of high-fructose corn syrup or bioethanol. Studying inhibitor effects on GI is important to deciphering GI-specific molecular functions, as well as potential industrial applications. Analysis of the existing xylitol-bound GI structure revealed low metal occupancy at the M2 site; however, it remains unknown why this phenomenon occurs. This study reports the room-temperature structures of native and xylitol-bound GI from Streptomyces rubiginosus (SruGI) determined by serial millisecond crystallography. The M1 site of native SruGI exhibits distorted octahedral coordination; however, xylitol binding results in the M1 site exhibit geometrically stable octahedral coordination. This change results in the rearrangement of metal-binding residues for the M1 and M2 sites, the latter of which previously displayed distorted metal coordination, resulting in unstable coordination of Mg2+ at the M2 site and possibly explaining the inducement of low metal-binding affinity. These results enhance the understanding of the configuration of the xylitol-bound state of SruGI and provide insights into its future industrial application. [ABSTRACT FROM AUTHOR]

Siderophores acquire iron from hosts under iron-limiting conditions and play an essential role in the survival of microorganisms. Siderophore-interacting proteins (SIPs) from microbes release iron from the siderophore complex by reducing ferric iron to ferrous iron, but the molecular mechanism of iron reduction remains unclear. To better understand the molecular mechanism of SIPs, we herein report the crystal structure of Aeromonas hydrophila SIP (AhSIP) in complex with flavin adenine dinucleotide (FAD) as a cofactor. AhSIP consists of an N-terminal FAD binding domain and a C-terminal NADH binding domain, which are connected by a linker region. AhSIP showed unique structural differences in the orientation of the cofactor binding lobes when compared with SIP homologs. This study identified a cluster of three basic residues (Lys48, His259 and Arg262) in AhSIP distributed around a potential substrate binding pocket. In addition, AhSIP, containing the NADH binding motif E(L)VL-X 3 -GE, belongs to the group I subfamily. Our results show the diverse cofactor and substrate binding sites of the SIP family. • Crystal structure determination of the Siderophore-interacting protein SIP-FAD complex from A. hydrophila. • AhSIP consist of N-terminal FAD binding domain and C-terminal NADH binding domain. • The potential substrate binding pocket of AhSIP and putative NADH binding sites are proposed. [ABSTRACT FROM AUTHOR]

Thiamin pyrophosphate (TPP) is an essential co-factor in amino acid and carbohydrate metabolic pathways. The TPP-related vitamin B1 biosynthetic pathway is found in most bacterial, plant and lower eukaryotic processes; however, it is not present in humans. In bacterial thiamin synthesis and salvage pathways, the 5-(hydroxyethyl)-methylthiazole kinase (ThiM) is essential in the pathway forming TPP. Thus, ThiM is considered to be an attractive antibacterial drug target. Here, we determined the crystal structures of ThiM from pathogenic Klebsiella pneumoniae (KpThiM) and KpThiM in complex with its substrate 5-(hydroxyethyl)-4-methylthiazole (TZE). KpThiM, consisting of an α-β-α domain, shows a pseudosymmetric trimeric formation. TZE molecules are located in the interface between the KpThiM subunits in the trimer and interact with Met49 and Cys200. Superimposition of the apo and TZE-complexed structures of KpThiM show that the side chains of the amino acids interacting with TZE and Mg2+ have a rigid configuration. Comparison of the ThiM structures shows that KpThiM could, in terms of sequence and configuration, be different from other ThiM proteins, which possess different amino acids that recognize TZE and Mg2+. The structures will provide new insight into the ThiM subfamily proteins for antibacterial drug development. • Crystal structure determination of ThiM-APO and ThiM-TZE complex from K. pneumonia. • The substrate binding site is located in the interface between the KpThiM subunits formed by the trimer.. • The substrate binding site and the magnesium ion binding site change of KpThiM are different from its homologue. • In bacterial thiamin synthesis and salvage pathways, ThiM is considered to be attractive antibacterial drug target. [ABSTRACT FROM AUTHOR]