Several Archaeal Homologs of Putative Oligopeptide-Binding Proteins Encoded byThermotoga maritimaBind Sugars

Abstract

The hyperthermophilic bacterium Thermotoga maritima has shared many genes with archaea through horizontal gene transfer. Several of these encode putative oligopeptide ATP binding cassette (ABC) transporters. We sought to test the hypothesis that these transporters actually transport sugars by measuring the substrate affinities of their encoded substrate-binding proteins (SBPs). This information will increase our understanding of the selective pressures that allowed this organism to retain these archaeal homologs. By measuring changes in intrinsic fluorescence of these SBPs in response to exposure to various sugars, we found that five of the eight proteins examined bind to sugars. We could not identify the ligands of the SBPs TM0460, TM1150, and TM1199. The ligands for the archaeal SBPs are TM0031 (BglE), the ␤-glucosides cellobiose and laminaribiose; TM0071 (XloE), xylobiose and xylotriose; TM0300 (GloE), large glucose oligosaccharides represented by xyloglucans; TM1223 (ManE), ␤-1,4-mannobiose; and TM1226 (ManD), ␤-1,4-mannobiose, ␤-1,4-mannotriose, ␤-1,4-mannotetraose, ␤-1,4-galactosyl mannobiose, and cellobiose. For comparison, seven bacterial putative sugar-binding proteins were examined and ligands for three (TM0595, TM0810, and TM1855) were not identified. The ligands for these bacterial SBPs are TM0114 (XylE), xylose; TM0418 (InoE), myo-inositol; TM0432 (AguE), ␣-1,4-digalactouronic acid; and TM0958 (RbsB), ribose. We found that T. maritima does not grow on several complex polypeptide mixtures as sole sources of carbon and nitrogen, so it is unlikely that these archaeal ABC transporters are used primarily for oligopeptide transport. Since these SBPs bind oligosaccharides with micromolar to nanomolar affinities, we propose that they are used primarily for oligosaccharide transport.

Insights into thermoadaptation and the evolution of mesophily from the bacterial phylum Thermotogae

“…Carbohydrate utilization by T. maritima has been examined by studying the substrate specificities and affinities of its carbohydrate transporters (Nanavati et al 2005(Nanavati et al , 2006Cuneo et al 2009;Boucher and Noll 2011;Ghimire-Rijal et al 2014) and their transcriptional regulation in response to growth on different saccharides (Frock et al 2012). Information about substrate specificities, enzymatic activities, and catalytic mechanisms of many of T. maritima's glycoside hydrolases are also available (Kleine and Liebl 2006;Comfort et al 2007;Arti et al 2012), which has been used, for instance, to engineer an ␣-galactosidase from T. maritima into an efficient ␣-galactosynthase (Cobucci-Ponzano et al 2011).…”

Structural Analysis of Semi-specific Oligosaccharide Recognition by a Cellulose-binding Protein of Thermotoga maritima Reveals Adaptations for Functional Diversification of the Oligopeptide Periplasmic Binding Protein Fold

“…It has been difficult to assign function reliably to the PBP components of these because of the distant sequence relationships between T. maritima and biochemically characterized model organisms. A combination of in vitro binding studies (25) and transcriptional expression profiling of cultures grown in the presence of various carbohydrate substrates (26,27) has shown that 5 of 12 PBPs that are homologous to OpBPs bind not oligopeptides but various carbohydrates. The x-ray crystal structure of one of these, the ␤-1,4-mannobiose-binding protein (Protein Data Bank code 1VR5), was recently solved in the open conformation in the absence of its cognate ligand (structure determined and deposited by the Joint Center for Structural Genomics (28) but otherwise undocumented).…”

“…To answer these questions, we undertook structural studies of tm0031, which was shown previously to bind the ␤-linked disaccharides cellobiose and laminaribiose (25). In additional ligand-binding studies, we established that this cellobiosebinding protein (tmCBP) binds not only disaccharides but also oligosaccharides up to five sugar monomers in length.…”

Functional Differences between Heme Permeases: Serratia marcescens HemTUV Permease Exhibits a Narrower Substrate Specificity (Restricted to Heme) Than the Escherichia coli DppABCDF Peptide-Heme Permease

“…Heme-loaded NikA is not involved in heme uptake in E. coli, but it might be involved in cytochrome maturation (18). This class is related to the Opp-like transporters, which are known to be involved in the transport of a large variety of substrates, such as sugar in archaea and thermophilic bacteria (12), proline betaine in Rhizobiaceae (2), and agrocinopine in Agrobacterium tumefaciens (6). The second class of periplasmic heme binding proteins includes smaller proteins, such as S. marcescens HemT, S. dysenteriae ShuT (3), and P. aeruginosa PhuT (23).…”