Enzymology
Mechanistic Strategies for Catalysis Adopted by Evolutionary Distinct Family 43 Arabinanases*

https://doi.org/10.1074/jbc.M113.537167Get rights and content
Under a Creative Commons license
open access

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. 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 Arg203–Ala230 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.

Biotechnology
Enzyme Kinetics
Glycoside Hydrolases
Protein Chimeras
Protein Structure
GH43 Family
Accessory Domain
Activation Mechanism
Arabinanase
Endo/Exo Activities

Cited by (0)

*

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Grants 10/51890-8 and 13/13309-0, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Grants 476043/2011-5 and 308092/2012-0, and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

1

Both authors contributed equally to this work.

3

The abbreviations used are:

    ABN

    arabinanase

    GH

    glycoside hydrolase

    ABF

    arabinofuranosidase

    TpABNΔC

    TpABN devoid of the accessory domain

    TRX

    thioredoxin.