Structural investigation of the transmembrane C domain of the mannitol permease from Escherichia coli using 5-FTrp fluorescence spectroscopy

Abstract

The mannitol transporter EII(mtl) from Escherichia coli is responsible for the uptake of mannitol over the inner membrane and its concomitant phosphorylation. EII(mtl) is functional as a dimer and its membrane-embedded C domain, IIC(mtl), harbors one high affinity mannitol binding site. To characterize this domain in more detail the microenvironments of thirteen residue positions were explored by 5-fluorotryptophan (5-FTrp) fluorescence spectroscopy. Because of the simpler photophysics of 5-FTrp compared to Trp, one can distinguish between the two 5-FTrp probes present in dimeric IIC(mtl). At many labeled positions, the microenvironment of the 5-FTrps in the two protomers differs. Spectroscopic properties of three mutants labeled at positions 198, 251, and 260 show that two conserved motifs (Asn194-His195 and Gly254-Ile255-His256-Glu257) are located in well-structured parts of IIC(mtl). Mannitol binding has a large impact on the structure around position 198, while only minor changes are induced at positions 251 and 260. Phosphorylation of the cytoplasmic B domain of EII(mtl) is sensed by 5-FTrp at positions 30, 42, 251 and 260. We conclude that many parts of the IIC(mtl) structure are involved in the sugar translocation. The structure of EII(mtl), as investigated in this work, differs from the recently solved structure of a IIC protein transporting diacetylchitobiose, ChbC, and also belonging to the glucose superfamily of EII sugar transporters. In EII(mtl), the sugar binding site is more close to the periplasmic face and the structure of the 2 protomers in the dimer is different, while both protomers in the ChbC dimer are essentially the same.

Characterization of D-Arabitol as Newly Discovered Carbon Source of Bacillus methanolicus

“…The finding that B. methanolicus MGA3 is able to catabolize low arabitol concentrations effectively ( K S value of about 3 mM) is in line with our prediction that arabitol is taken up by the cells via arabitol PTS encoded by arabitol inducible genes atlABC . High substrate affinity has been reported in several organisms in relation to PTS-mediated uptake (Nothaft et al, 2003; Lindner et al, 2011; Opačić et al, 2012), as is also the case for mannitol in B. methanolicus : the K S value for mannitol was determined to be 0.2 ± 0.1 mM with genes mtlF, mtlA , and mtlD known to code for a mannitol-specific PTS enzyme IIA component, a mannitol-specific PTS enzyme IIBC component and a mannitol-1-phosphate 5-dehydrogenase, respectively (Heggeset et al, 2012; Irla et al, 2016). However, the observed growth rates and substrate uptake rates were higher for mannitol than for arabitol, and the biomass yield for the C5 sugar alcohol arabitol was lower than for the C6 sugar alcohol mannitol (Table 2).…”

Structural insight into the PTS sugar transporter EIIC

“…There is currently no evidence of cooperativity in the bcChbC dimer; however, given that the cytoplasmic gate appears to be supplied by the opposing protomer, it is possible that this could provide a mechanism for cooperativity, as sugar exiting the cavity of one protomer could be sensed by the other. Furthermore, residues in the N-terminal half of MtlA (the region corresponding from TM1 to TM4 in bcCHBC) were shown to be in close contact with mannitol, the sugar was shown to be located closer to the periplasm, and substantial differences were observed between solvent exposed residues in MtlA and their expected counterparts in the bcChbc structure[69, 70]. Tryptophan phosphorescence spectroscopy indicated that F97 in MtlA (a predicted cytoplasmic loop residue that overlays TM3 in bcChbC) is part of a β-sheet fold[71].…”

Identification of resistance in Escherichia coli and Klebsiella pneumoniae using excitation-emission matrix fluorescence spectroscopy and multivariate analysis

“…Satisfactory results from the models using EEM fluorescence data, for the E. coli and K. pneumoniae bacteria, demonstrate the sensitivity of the technique in detecting variations in the nuclear content of the cells and in the structure of the membranes itself. As reported by Opačić et al 19 , who used fluorescence spectroscopy on structural investigation of the transmembrane C domain of the mannitol permease from Escherichia coli, the results showed that the technique was capable to differentiated the structure of EII mtl from structure of a IIC protein transporting diacetylchitobiose. Additionally, Romantsov et.…”

“…As reported by Opačić et al . 19 , who used fluorescence spectroscopy on structural investigation of the transmembrane C domain of the mannitol permease from Escherichia coli , the results showed that the technique was capable to differentiated the structure of EII mtl from structure of a IIC protein transporting diacetylchitobiose. Additionally, Romantsov et.…”

“…Fluorescence spectroscopy has already been used in the detection 18 , structural investigation 19 , 20 and in the construction of a DNA biosensor for E. coli 21 . Chemometric methods such as Linear Discriminant Analysis (LDA) 22 , Quadratic Discriminant Analysis (QDA) 23 and Support Vector Machines (SVM) 24 , coupled with the dimensionality reduction algorithm: Principal Component Analysis (PCA) 25 , 26 ,and variable selection algorithms: Genetic Algorithm (GA) 27 and Successive Projections Algorithm (SPA) 28 , tend to enhance the spectroscopic techniques 29 – 31 .…”