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An evolutionarily distinct family of polysaccharide lyases removes rhamnose capping of complex arabinogalactan proteins.

ABSTRACT: The human gut microbiota utilizes complex carbohydrates as major nutrients. The requirement for efficient glycan degrading systems exerts a major selection pressure on this microbial community. Thus, we propose that this microbial ecosystem represents a substantial resource for discovering novel carbohydrate active enzymes. To test this hypothesis we screened the potential enzymatic functions of hypothetical proteins encoded by genes of Bacteroides thetaiotaomicron that were up-regulated by arabinogalactan proteins or AGPs. Although AGPs are ubiquitous in plants, there is a paucity of information on their detailed structure, the function of these glycans in planta, and the mechanisms by which they are depolymerized in microbial ecosystems. Here we have discovered a new polysaccharide lyase family that is specific for the l-rhamnose-?1,4-d-glucuronic acid linkage that caps the side chains of complex AGPs. The reaction product generated by the lyase, ?4,5-unsaturated uronic acid, is removed from AGP by a glycoside hydrolase located in family GH105, producing the final product 4-deoxy-?-l-threo-hex-4-enepyranosyl-uronic acid. The crystal structure of a member of the novel lyase family revealed a catalytic domain that displays an (?/?)6 barrel-fold. In the center of the barrel is a deep pocket, which, based on mutagenesis data and amino acid conservation, comprises the active site of the lyase. A tyrosine is the proposed catalytic base in the ?-elimination reaction. This study illustrates how highly complex glycans can be used as a scaffold to discover new enzyme families within microbial ecosystems where carbohydrate metabolism is a major evolutionary driver.

SUBMITTER: Munoz-Munoz J

PROVIDER: S-EPMC5555188 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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An evolutionarily distinct family of polysaccharide lyases removes rhamnose capping of complex arabinogalactan proteins.

Munoz-Munoz José J Cartmell Alan A Terrapon Nicolas N Baslé Arnaud A Henrissat Bernard B Gilbert Harry J HJ

The Journal of biological chemistry 20170621 32

The human gut microbiota utilizes complex carbohydrates as major nutrients. The requirement for efficient glycan degrading systems exerts a major selection pressure on this microbial community. Thus, we propose that this microbial ecosystem represents a substantial resource for discovering novel carbohydrate active enzymes. To test this hypothesis we screened the potential enzymatic functions of hypothetical proteins encoded by genes of <i>Bacteroides thetaiotaomicron</i> that were up-regulated ...[more]

PMID: 28637865

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Project description:Members of the Ets family of proto-oncogenes encode sequence-specific transcription factors that bind to a purine-rich motif centered around a conserved GGA trinucleotide. Ets binding sites have been identified in the transcriptional regulatory regions of multiple T cell genes including the T cell receptor alpha and beta (TCR-alpha and -beta) enhancers and the IL-2 enhancer, as well as in the enhancers of several T cell-trophic viruses including Maloney sarcoma virus, human leukemia virus type 1, and human immunodeficiency virus-2. T cells express multiple members of the Ets gene family including Ets-1, Ets-2, GABP alpha, Elf-1, and Fli-1. The different patterns of expression and protein-protein interactions of these different Ets family members undoubtedly contribute to their ability to specifically regulate distinct sets of T cell genes. However, previous studies have suggested that different Ets family members might also display distinct DNA binding specificities. In this report, we have examined the DNA binding characteristics of two Ets family members, Ets-1 and Elf-1, that are highly expressed in T cells. The results demonstrate that the minimal DNA binding domain of these proteins consists of adjacent basic and putative alpha-helical regions that are conserved in all of the known Ets family members. Both regions are required for DNA binding activity. In vitro binding studies demonstrated that Ets-1 and Elf-1 display distinct DNA binding specificities, and, thereby interact preferentially with different naturally occurring Ets binding sites. A comparison of known Ets binding sites identified three nucleotides at the 3' end of these sequences that control the differential binding of the Ets-1 and Elf-1 proteins. These results are consistent with a model in which different Ets family members regulate the expression of different T cell genes by binding preferentially to purine-rich sequences that share a GGA core motif, but contain distinct flanking sequences.

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Dataset Information

An evolutionarily distinct family of polysaccharide lyases removes rhamnose capping of complex arabinogalactan proteins.

Publications

An evolutionarily distinct family of polysaccharide lyases removes rhamnose capping of complex arabinogalactan proteins.

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