Herman van Tilbeurgh

Limited proteolysis of the cellobiohydrolase I from Trichoderma reesei: Separation of functional domains

Limited proteolysis of the cellobiohydrolase I (CBH I, 65 kDa) from Trichoderma reesei by papain yields a core protein (56 kDa) which is fully active against small, soluble substrates such as the chromophoric glycosides derived from the cellodextrins and lactose. Activity against an insoluble substrate, such as Avicel, is however completely lost and concomitantly decreased adsorption onto this microcrystalline cellulose is observed. The peptide (10 kDa), initially split off during proteolysis, is identified as the heavily glycosylated carboxy‐terminal of the native CBH I. Depending on the experimental conditions the core protein is further nicked in between disulfide bonds, but its properties and stability do not appreciably differ from those of intact CBH I. These results lead to the proposal of a bifunctional organisation of the CBH I: one domain, corresponding to the carboxyterminal, acts as a binding site for insoluble cellulose and the other, localised in the core protein, contains the active (hydrolytic) site.

The use of 4-methylumbelliferyl and other chromophoric glycosides in the study of cellulolytic enzymes

HPLC‐analysis of the reaction products of a series of 4‐methylumbelliferyl glycosides from cello‐oligosaccharides, used as substrates of a cellobiohydrolase from Trichoderma reesei, proves the lack of specificity for terminal cellobiosyl groups. Also, different reaction patterns are observed for this CBHI and for an endocellulase, when acting on these same substrates. 4‐Methylumbelliferyl β‐D‐lactoside is an unexpected substrate for CBHI, yielding only lactose and phenol as reaction products. The binding characteristics of p‐nitrobenzyl 1‐thio‐β‐D‐lactoside for this enzyme are determined by a dia‐filtration technique, yielding 1 binding site and an association constant of 4.0 × 104 M−1.

Structural basis for the substrate selectivity of pancreatic lipases and some related proteins

The classical human pancreatic lipase (HPL), the guinea pig pancreatic lipase-related protein 2 (GPLRP2) and the phospholipase A1 from hornet venom (DolmI PLA1) illustrate three interesting steps in the molecular evolution of the pancreatic lipase gene family towards different substrate selectivities. Based on the known 3D structures of HPL and a GPLRP2 chimera, as well as the modeling of DolmI PLA1, we review here the structural features and the kinetic properties of these three enzymes for a better understanding of their structure-function relationships. HPL displays significant activity only on triglycerides, whereas GPLRP2 displays high phospholipase and galactolipase activities, together with a comparable lipase activity. GPLRP2 shows high structural homology with HPL with the exception of the lid domain which is made of five amino acid residues (mini-lid) instead of 23 in HPL. The lid domain deletion in GPLRP2 allows the free access to the active site and reduces the steric hindrance towards large substrates, such as galactolipids. The role of the lid domain in substrate selectivity has been investigated by site-directed mutagenesis and the substitution of HPL and GPLRP2 lid domains. The addition of a large-size lid domain in GPLRP2 increases the substrate selectivity for triglycerides by depressing the phospholipase activity. The phospholipase activity is, however, not induced in the case of the HPL mutant with GPLRP2 mini-lid. Therefore, the presence of a full-length lid domain is not the unique structural feature explaining the absence of phospholipase activity in HPL. The 3D structure of the GPLRP2 chimera and the model of DolmI PLA1 reveal a higher hydrophilic/lipophilic balance (HLB) of the surface loops (beta5 loop, beta9 loop, lid domain) surrounding the active site, as compared to the homologous loops in HPL. This observation provides a potential explanation for the ability of GPLRP2 and DolmI PLA1 to hydrolyze polar lipids, such as phospholipids. In conclusion, the beta5 loop, the beta9 loop, and the lid domain play an essential role in substrate selectivity towards triglycerides, phospholipids and galactolipids.

Crystal Structure of Maltose Phosphorylase from Lactobacillus brevis: Unexpected Evolutionary Relationship with Glucoamylases

BACKGROUND Maltose phosphorylase (MP) is a dimeric enzyme that catalyzes the conversion of maltose and inorganic phosphate into beta-D-glucose-1-phosphate and glucose without requiring any cofactors, such as pyridoxal phosphate. The enzyme is part of operons that are involved in maltose/malto-oligosaccharide metabolism. Maltose phosphorylases have been classified in family 65 of the glycoside hydrolases. No structure is available for any member of this family. RESULTS We report here the 2.15 A resolution crystal structure of the MP from Lactobacillus brevis in complex with the cosubstrate phosphate. This represents the first structure of a disaccharide phosphorylase. The structure consists of an N-terminal complex beta sandwich domain, a helical linker, an (alpha/alpha)6 barrel catalytic domain, and a C-terminal beta sheet domain. The (alpha/alpha)6 barrel has an unexpected strong structural and functional analogy with the catalytic domain of glucoamylase from Aspergillus awamori. The only conserved glutamate of MP (Glu487) superposes onto the catalytic residue Glu179 of glucoamylase and likely represents the general acid catalyst. The phosphate ion is bound in a pocket facing the carboxylate of Glu487 and is ideally positioned for nucleophilic attack of the anomeric carbon atom. This site is occupied by the catalytic base carboxylate in glucoamylase. CONCLUSIONS These observations strongly suggest that maltose phosphorylase has evolved from glucoamylase. MP has probably conserved one carboxylate group for acid catalysis and has exchanged the catalytic base for a phosphate binding pocket. The relative positions of the acid catalytic group and the bound phosphate are compatible with a direct-attack mechanism of a glycosidic bond by phosphate, in accordance with inversion of configuration at the anomeric carbon as observed for this enzyme.

Prp43p contains a processive helicase structural architecture with a specific regulatory domain

The DEAH/RNA helicase A (RHA) helicase family comprises proteins involved in splicing, ribosome biogenesis and transcription regulation. We report the structure of yeast Prp43p, a DEAH/RHA helicase remarkable in that it functions in both splicing and ribosome biogenesis. Prp43p displays a novel structural architecture with an unforeseen homology with the Ski2‐like Hel308 DNA helicase. Together with the presence of a β‐hairpin in the second RecA‐like domain, Prp43p contains all the structural elements of a processive helicase. Moreover, our structure reveals that the C‐terminal domain contains an oligonucleotide/oligosaccharide‐binding (OB)‐fold placed at the entrance of the putative nucleic acid cavity. Deletion or mutations of this domain decrease the affinity of Prp43p for RNA and severely reduce Prp43p ATPase activity in the presence of RNA. We also show that this domain constitutes the binding site for the G‐patch‐containing domain of Pfa1p. We propose that the C‐terminal domain, specific to DEAH/RHA helicases, is a central player in the regulation of helicase activity by binding both RNA and G‐patch domain proteins.

Fluorogenic and chromogenic glycosides as substrates and ligands of carbohydrases

Publisher Summary Nitrophenylglycosides are frequently used substrates of carbohydrases. Alternatively, the 4-methylumbelliferyl (7-hydroxy-4-methylcoumaryl) derivatives offer a more sensitive (fluorometric) method of detection. Some of these compounds have become commercially available. This chapter describes the preparation and use of these glycosides in the study of some cellulolytic enzymes. The difference in spectral properties of free 4-methylumbelliferone and its carbohydrate conjugates allows sensitive and continuous assays of cellulolytic activities in absorption or fluorescence modes. The preparation and use of 1-thio derivatives with different chromophoric reporter groups are included. Due to their optical characteristics, the chromophoric (fluorochromic) derivatives offer distinct advantages over the use of classical substrates of cellulolytic enzymes, as they are sensitive tools for the determination of the number of binding sites, study of association modes, and binding kinetics, breakdown patterns, and inhibition characteristics. The chapter describes some applications of chromophoric.

Structural inhibition of the colicin D tRNase by the tRNA‐mimicking immunity protein

Colicins are toxins secreted by Escherichia coli in order to kill their competitors. Colicin D is a 75 kDa protein that consists of a translocation domain, a receptor‐binding domain and a cytotoxic domain, which specifically cleaves the anticodon loop of all four tRNAArg isoacceptors, thereby inactivating protein synthesis and leading to cell death. Here we report the 2.0 Å resolution crystal structure of the complex between the toxic domain and its immunity protein ImmD. Neither component shows structural homology to known RNases or their inhibitors. In contrast to other characterized colicin nuclease–Imm complexes, the colicin D active site pocket is completely blocked by ImmD, which, by bringing a negatively charged cluster in opposition to a positively charged cluster on the surface of colicin D, appears to mimic the tRNA substrate backbone. Site‐directed mutations affecting either the catalytic domain or the ImmD protein have led to the identification of the residues vital for catalytic activity and for the tight colicin D/ImmD interaction that inhibits colicin D toxicity and tRNase catalytic activity.

Structure of Yeast Dom34 : A PROTEIN RELATED TO TRANSLATION TERMINATION FACTOR Erf1 AND INVOLVED IN No-Go DECAY

The yeast protein Dom34 has been described to play a critical role in a newly identified mRNA decay pathway called No-Go decay. This pathway clears cells from mRNAs inducing translational stalls through endonucleolytic cleavage. Dom34 is related to the translation termination factor eRF1 and physically interacts with Hbs1, which is itself related to eRF3. We have solved the 2.5-Å resolution crystal structure of Saccharomyces cerevisiae Dom34. This protein is organized in three domains with the central and C-terminal domains structurally homologous to those from eRF1. The N-terminal domain of Dom34 is different from eRF1. It adopts a Sm-fold that is often involved in the recognition of mRNA stem loops or in the recruitment of mRNA degradation machinery. The comparison of eRF1 and Dom34 domains proposed to interact directly with eRF3 and Hbs1, respectively, highlights striking structural similarities with eRF1 motifs identified to be crucial for the binding to eRF3. In addition, as observed for eRF1 that enhances eRF3 binding to GTP, the interaction of Dom34 with Hbs1 results in an increase in the affinity constant of Hbs1 for GTP but not GDP. Taken together, these results emphasize that eukaryotic cells have evolved two structurally related complexes able to interact with ribosomes either paused at a stop codon or stalled in translation by the presence of a stable stem loop and to trigger ribosome release by catalyzing chemical bond hydrolysis.

Protein-carbohydrate interactions

Abstract X-ray structures of protein-carbohydrate complexes reported this year confirm well established rules of protein-carbohydrate interactions: stabilization through a network of hydrogen bonds often involving water molecules, and hydrophobic interactions. The importance of the latter interactions have been tested in a few cases by site-directed mutagenesis experiments. In contrast, new insights have emerged regarding biologically and catalytically relevant carbohydrate-metal interactions. Carbohydrate flexibility and ring opening play crucial roles in catalysis, as demonstrated by the structures of neuraminidase and xylose isomerase.

Structural insights into the regulation of bacterial signalling proteins containing PRDs

PRD-containing proteins are bacterial transcriptional antiterminators and activators characterised by a duplicated phosphorylation domain involved in the regulation of catabolic operons. Recent genetic and biochemical studies have suggested how the activity of these regulators is positively or negatively controlled through the multiple phosphorylation of conserved histidyl residues. The regulation mode of these proteins has been examined in light of the recently determined first crystal structure of the phosphorylatable domain of the LicT antiterminator.