Dataset Information

Mechanistic strategies for catalysis adopted by evolutionary distinct family 43 arabinanases.

ABSTRACT: Arabinanases (ABNs, EC 3.2.1.99) are promising catalysts for environmentally friendly biomass conversion into energy and chemicals. These enzymes catalyze the hydrolysis of the ?-1,5-linked L-arabinofuranoside backbone of plant cell wall arabinans releasing arabino-oligosaccharides and arabinose, the second most abundant pentose in nature. In this work, new findings about the molecular mechanisms governing activation, functional differentiation, and catalysis of GH43 ABNs are presented. Biophysical, mutational, and biochemical studies with the hyperthermostable two-domain endo-acting ABN from Thermotoga petrophila (TpABN) revealed how some GH43 ABNs are activated by calcium ions via hyperpolarization of the catalytically relevant histidine and the importance of the ancillary domain for catalysis and conformational stability. On the other hand, the two GH43 ABNs from rumen metagenome, ARN2 and ARN3, presented a calcium-independent mechanism in which sodium is the most likely substituent for calcium ions. The crystal structure of the two-domain endo-acting ARN2 showed that its ability to efficiently degrade branched substrates is due to a larger catalytic interface with higher accessibility than that observed in other ABNs with preference for linear arabinan. Moreover, crystallographic characterization of the single-domain exo-acting ARN3 indicated that its cleavage pattern producing arabinose is associated with the chemical recognition of the reducing end of the substrate imposed by steric impediments at the aglycone-binding site. By structure-guided rational design, ARN3 was converted into a classical endo enzyme, confirming the role of the extended Arg(203)-Ala(230) loop in determining its action mode. These results reveal novel molecular aspects concerning the functioning of GH43 ABNs and provide new strategies for arabinan degradation.

SUBMITTER: Santos CR

PROVIDER: S-EPMC3953252 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Mechanistic strategies for catalysis adopted by evolutionary distinct family 43 arabinanases.

Santos Camila R CR Polo Carla C CC Costa Maria C M F MC Nascimento Andrey F Z AF Meza Andreia N AN Cota Junio J Hoffmam Zaira B ZB Honorato Rodrigo V RV Oliveira Paulo S L PS Goldman Gustavo H GH Gilbert Harry J HJ Prade Rolf A RA Ruller Roberto R Ruller Roberto R Squina Fabio M FM Wong Dominic W S DW Murakami Mário T MT

The Journal of biological chemistry 20140127 11

Arabinanases (ABNs, EC 3.2.1.99) are promising catalysts for environmentally friendly biomass conversion into energy and chemicals. These enzymes catalyze the hydrolysis of the α-1,5-linked L-arabinofuranoside backbone of plant cell wall arabinans releasing arabino-oligosaccharides and arabinose, the second most abundant pentose in nature. In this work, new findings about the molecular mechanisms governing activation, functional differentiation, and catalysis of GH43 ABNs are presented. Biophysi ...[more]

PMID: 24469445

Similar Datasets

Project description:Resistance genes play an important role in the defense of plants against the invasion of pathogens. In Setaria italica and closely related grass species, R genes have been identified through genetic mapping and genome-wide homologous/domain searching. However, there has been to date no systematic analysis of the evolutionary features of R genes across all sequenced grass genomes. Here, we determined and comprehensively compared R genes in all 12 assembled grass genomes and an outgroup species (Arabidopsis thaliana) through synteny and selection analyses of multiple genomes. We found that the two groups of nucleotide binding site (NBS) domains containing R genes-R tandem duplications (TD) and R singletons-adopted different strategies and showed different features in their evolution. Based on Ka/Ks analysis between syntenic R loci pairs of TDs or singletons, we conclude that R singletons are under stronger purifying selection to be conserved among different grass species than R TDs, while R genes located at TD arrays have evolved much faster through diversifying selection. Furthermore, using the variome datasets of S. italica populations, we scanned for selection signals on genes and observed that a part of R singleton genes have been under purifying selection in populations of S. italica, which is consistent with the pattern observed in syntenic R singletons among different grass species. Additionally, we checked the synteny relationships of reported R genes in grass species and found that the functionally mapped R genes for novel resistance traits are prone to appear in TDs and are heavily divergent from their syntenic orthologs in other grass species, such the black streak R gene Rxo1 in Z. mays and the blast R gene Pi37 in O. sativa. These findings indicate that the R genes from TDs adopted tandem duplications to evolve faster and accumulate more mutations to facilitate functional innovation to cope with variable threats from a fluctuating environment, while R singletons provide a way for R genes to maintain sequence stability and retain conservation of function.

Project description:Ulvan is an important marine polysaccharide. Bacterial ulvan lyases play important roles in ulvan degradation and marine carbon cycling. Until now, only a small number of ulvan lyases have been characterized. Here, a new ulvan lyase, Uly1, belonging to polysaccharide lyase family 24 (PL24) from the marine bacterium Catenovulum maritimum, is characterized. The optimal temperature and pH for Uly1 to degrade ulvan are 40°C and pH 9.0, respectively. Uly1 degrades ulvan polysaccharides in the endolytic manner, mainly producing ΔRha3S, consisting of an unsaturated 4-deoxy-l-threo-hex-4-enopyranosiduronic acid and a 3-O-sulfated α-l-rhamnose. The structure of Uly1 was resolved at a 2.10-Å resolution. Uly1 adopts a seven-bladed β-propeller architecture. Structural and site-directed mutagenesis analyses indicate that four highly conserved residues, H128, H149, Y223, and R239, are essential for catalysis. H128 functions as both the catalytic acid and base, H149 and R239 function as the neutralizers, and Y223 plays a supporting role in catalysis. Structural comparison and sequence alignment suggest that Uly1 and many other PL24 enzymes may directly bind the substrate near the catalytic residues for catalysis, different from the PL24 ulvan lyase LOR_107, which adopts a two-stage substrate binding process. This study provides new insights into ulvan lyases and ulvan degradation. IMPORTANCE Ulvan is a major cell wall component of green algae of the genus Ulva. Many marine heterotrophic bacteria can produce extracellular ulvan lyases to degrade ulvan for a carbon nutrient. In addition, ulvan has a range of physiological bioactivities based on its specific chemical structure. Ulvan lyase thus plays an important role in marine carbon cycling and has great potential in biotechnological applications. However, only a small number of ulvan lyases have been characterized over the past 10 years. Here, based on biochemical and structural analyses, a new ulvan lyase of polysaccharide lyase family 24 is characterized, and its substrate recognition and catalytic mechanisms are revealed. Moreover, a new substrate binding process adopted by PL24 ulvan lyases is proposed. This study offers a better understanding of bacterial ulvan lyases and is helpful for studying the application potentials of ulvan lyases.

Dataset Information

Mechanistic strategies for catalysis adopted by evolutionary distinct family 43 arabinanases.

Publications

Mechanistic strategies for catalysis adopted by evolutionary distinct family 43 arabinanases.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets