RESUMO
Recent metagenomic analyses have identified uncultured bacteria that are abundant in the rumen of herbivores and that possess putative biomass-converting enzyme systems. Here we investigate the saccharolytic capabilities of a polysaccharide utilization locus (PUL) that has been reconstructed from an uncultured Bacteroidetes phylotype (SRM-1) that dominates the rumen microbiome of Arctic reindeer. Characterization of the three PUL-encoded outer membrane glycoside hydrolases was performed using chromogenic substrates for initial screening, followed by detailed analyses of products generated from selected substrates, using high-pressure anion-exchange chromatography with electrochemical detection. Two glycoside hydrolase family 5 (GH5) endoglucanases (GH5_g and GH5_h) demonstrated activity against ß-glucans, xylans, and xyloglucan, whereas GH5_h and the third enzyme, GH26_i, were active on several mannan substrates. Synergy experiments examining different combinations of the three enzymes demonstrated limited activity enhancement on individual substrates. Binding analysis of a SusE-positioned lipoprotein revealed an affinity toward ß-glucans and, to a lesser extent, mannan, but unlike the two SusD-like lipoproteins previously characterized from the same PUL, binding to cellulose was not observed. Overall, these activities and binding specificities correlated well with the glycan content of the reindeer rumen, which was determined using comprehensive microarray polymer profiling and showed an abundance of various hemicellulose glycans. The substrate versatility of this single PUL putatively expands our perceptions regarding PUL machineries, which so far have demonstrated gene organization that suggests one cognate PUL for each substrate type. The presence of a PUL that possesses saccharolytic activity against a mixture of abundantly available polysaccharides supports the dominance of SRM-1 in the Svalbard reindeer rumen microbiome.
Assuntos
Adaptação Biológica , Bacteroidetes/genética , Bacteroidetes/metabolismo , Redes e Vias Metabólicas , Polissacarídeos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Técnicas Eletroquímicas , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Metagenômica , Dados de Sequência Molecular , Ligação Proteica , Rena , Rúmen/microbiologia , Análise de Sequência de DNA , Especificidade por Substrato , SvalbardRESUMO
We demonstrate that two characteristic Sus-like proteins encoded within a polysaccharide utilization locus (PUL) bind strongly to cellulosic substrates and interact with plant primary cell walls. This shows associations between uncultured Bacteroidetes-affiliated lineages and cellulose in the rumen and thus presents new PUL-derived targets to pursue regarding plant biomass degradation.
Assuntos
Proteínas de Bactérias/metabolismo , Bacteroidetes/genética , Celulose/metabolismo , Animais , Arabidopsis/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Parede Celular/microbiologia , DNA Bacteriano/química , DNA Bacteriano/genética , Dados de Sequência Molecular , Ligação Proteica , Rúmen/microbiologia , Análise de Sequência de DNARESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Previous gene-centric analysis of a cow rumen metagenome revealed the first potentially cellulolytic polysaccharide utilization locus, of which the main catalytic enzyme (AC2aCel5A) was identified as a glycoside hydrolase (GH) family 5 endo-cellulase. Here we present the 1.8 Å three-dimensional structure of AC2aCel5A, and characterization of its enzymatic activities. The enzyme possesses the archetypical (ß/α)8-barrel found throughout the GH5 family, and contains the two strictly conserved catalytic glutamates located at the C-terminal ends of ß-strands 4 and 7. The enzyme is active on insoluble cellulose and acts exclusively on linear ß-(1,4)-linked glucans. Co-crystallization of a catalytically inactive mutant with substrate yielded a 2.4 Å structure showing cellotriose bound in the -3 to -1 subsites. Additional electron density was observed between Trp178 and Trp254, two residues that form a hydrophobic "clamp", potentially interacting with sugars at the +1 and +2 subsites. The enzyme's active-site cleft was narrower compared to the closest structural relatives, which in contrast to AC2aCel5A, are also active on xylans, mannans and/or xyloglucans. Interestingly, the structure and function of this enzyme seem adapted to less-substituted substrates such as cellulose, presumably due to the insufficient space to accommodate the side-chains of branched glucans in the active-site cleft.
Assuntos
Proteínas de Bactérias/química , Bacteroidetes/enzimologia , Celulase/química , Celulose/análogos & derivados , Celulose/química , Domínio Catalítico , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
Uncultured and therefore uncharacterized Bacteroidetes lineages are ubiquitous in many natural ecosystems which specialize in lignocellulose degradation. However, their metabolic contribution remains mysterious, as well-studied cultured Bacteroidetes have been shown to degrade only soluble polysaccharides within the human distal gut and herbivore rumen. We have interrogated a reconstructed genome from an uncultured Bacteroidetes phylotype that dominates a switchgrass-associated community within the cow rumen. Importantly, this characterization effort has revealed the first preliminary evidence for polysaccharide utilization locus (PUL)-catalyzed conversion of cellulose. Based on these findings, we propose a further expansion of the PUL paradigm and the saccharolytic capacity of rumen Bacteroidetes species to include cellulose, the most abundant terrestrial polysaccharide on Earth. Moreover, the perspective of a cellulolytic PUL lays the foundation for PULs to be considered an alternative mechanism for cellulose degradation, next to cellulosomes and free-enzyme systems.