To reveal the structural principles determining substrate specificity of 4-coumarate:CoA ligase (4CL), the crystal structure of the phenylalanine activation domain of gramicidin S synthetase was used as a template for homology modeling. According to our model, 12 amino acid residues lining the Arabidopsis 4CL isoform 2 (At4CL2) substrate binding pocket (SBP) function as a signature motif generally determining 4CL substrate specificity. We used this substrate specificity code to create At4CL2 gain-of-function mutants. By increasing the space within the SBP we generated ferulicand sinapic acid-activating At4CL2 variants. Increasing the hydrophobicity of the SBP resulted in At4CL2 variants with strongly enhanced conversion of cinnamic acid. These enzyme variants are suitable tools for investigating and influencing metabolic channeling mediated by 4CL. Knowledge of the 4CL specificity code will facilitate the prediction of substrate preference of numerous, still uncharacterized 4CL-like proteins.
contributed equally to this work High-resolution crystal structures of a-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycosidase, are described. The enzyme is a hexamer, and each monomer is organized into two domains: a (b/a) 8 -barrel and a 12-stranded b sandwich with jelly-roll topology. The structures of the Michaelis complexes with natural and synthetic substrates, and of the transient covalent arabinofuranosyl± enzyme intermediate represent two stable states in the double displacement mechanism, and allow thorough examination of the catalytic mechanism. The arabinofuranose sugar is tightly bound and distorted by an extensive network of hydrogen bonds. The two catalytic residues are 4.7 A Ê apart, and together with other conserved residues contribute to the stabilization of the oxocarbenium ion-like transition state via charge delocalization and speci®c protein±substrate interactions. The enzyme is an anti-protonator, and a 1.7 A Ê electrophilic migration of the anomeric carbon takes place during the hydrolysis. Keywords: arabinofuranosidase/clan GH-A glycosidase/ enzyme mechanism/glycoside hydrolase family 51/X-ray crystallography IntroductionMost of the biomass synthesized by photosynthetic CO 2 ®xation is stored in the plant cell wall as polymeric carbohydrates, mainly in the form of cellulose and hemicellulose. The degradation of these polymers is a key step in the carbon cycle, and is mediated by microorganisms that produce speci®c enzymes, i.e. cellulases and hemicellulases ( Figure 1A) . a-L-Arabinofuranosidases (EC 3.2.1.55) are hemicellulases that hydrolyze the arabinofuranosyl substitutions in hemicellulose. Some of these enzymes exhibit broad substrate speci®city, acting on arabinofuranoside moieties at O-2 or O-3 as a single substituent, as well as from O-2 and O-3 doubly substituted xylans, xylooligomers and a-1,5-linked arabinans (Saha, 2000). The glycosidic bond between two sugars is one of the most stable bonds in nature, and its enzymatic hydrolysis, carried out by glycoside hydrolases, provides an acceleration rate (k cat /k uncat ) which can be as high as 10 17 -fold (Wolfenden et al., 1998). The key elements in this remarkable catalysis are the ®nely tuned positions of the catalytic residues, and the distortion of the sugar ring so as to allow the stabilization of an oxocarbenium-ion-like transition state, as well as the movement of the hydrogen on the anomeric carbon to allow a direct nucleophilic attack (Zechel and Withers, 1999). Many glycosidases are modular proteins, and in addition to their catalytic domains include other functional modules, mainly carbohydrate-binding modules (CBMs) (Bourne and Henrissat, 2001). Currently, >8500 glycosidase sequences are known, and the sequence-based classi®cation of their catalytic domains into glycoside hydrolase (GH) families and clans is available on the continuously updated Carbohydrate-Active Enzymes (CAZy) server (http:// afmb.cnrs-mrs.fr/CAZY). The different bacterial, fungal and plant a-L-arabinofuranosidases...
Alpha-l-arabinofuranosidases (EC 3.2.1.55) are hemicellulases that cleave the glycosidic bond between l-arabinofuranoside side chains and various oligosaccharides. In this study, the first crystallization and preliminary X-ray analysis of the alpha-l-arabinofuranosidase from Geobacillus stearothermophilus T-6 (AbfA T-6), a family 51 glycoside hydrolase, is described. AbfA T-6 is a hexameric protein consisting of six identical subunits of 502 amino acids and with a calculated molecular mass of 57 218 Da. Purified recombinant native and selenomethionine-containing AbfA T-6 were crystallized by the sitting-drop method in two different space groups, P2(1) (unit-cell parameters a = 100.8, b = 178.1, c = 196.2 A, beta = 96.1 degrees ) and R3 (unit-cell parameters a = b = 179.3, c = 100.4 A). The R3 crystals diffracted X-rays to a resolution of 1.8 A.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.