TIM Barrel Domain Research Articles

Mechanisms of efficient polyacrylamide degradation: From multi-omics analysis to structural characterization of two amidohydrolases

Polyacrylamide (PAM) is a high molecular weight polymer with extensive applications. However, inefficient natural degradation of PAM results in its environmental accumulation. Here, using multi-omics analysis, we constructed the PAM biodegradation pathway in Klebsiella sp. PCX, an efficient PAM-degrading bacterium. Subsequently, two unclassified amidohydrolases (PCX00451 and PCX04581) were identified as key factors for rapid PAM biodegradation, both of which possessed much higher hydrolysis efficiency for PAM than for small molecule amide compounds. Besides, crystal structures of PCX00451 and PCX04581 were solved. Both two amidohydrolases were consisted with a twisted triosephosphateisomerase (TIM)-barrel and a smaller β-sandwich domain. And their binding pockets were in the conserved metal center of TIM-barrel domain. Moreover, Asp267 of PCX00451 and Asp282 of PCX04581 were examined as active sites for acid/base catalysis. Our research characterized the molecular mechanisms of two efficient amidohydrolases, providing theoretical basis and valuable tools for PAM bioremediation.

Reconstruction and identification of the native PLP synthase complex from Methanosarcina acetivorans lysate.

Many protein-protein interactions behave differently in biochemically purified forms as compared to their in vivo states. As such, determining native protein structures may elucidate structural states previously unknown for even well-characterized proteins. Here we apply the bottom-up structural proteomics method, cryoID , toward a model methanogenic archaeon. While they are keystone organisms in the global carbon cycle and active members of the human microbiome, there is a general lack of characterization of methanogen enzyme structure and function. Through the cryoID approach, we successfully reconstructed and identified the native Methanosarcina acetivorans pyridoxal 5'-phosphate (PLP) synthase (PdxS) complex directly from cryogenic electron microscopy (cryoEM) images of fractionated cellular lysate. We found that the native PdxS complex exists as a homo-dodecamer of PdxS subunits, and the previously proposed supracomplex containing both the synthase (PdxS) and glutaminase (PdxT) was not observed in cellular lysate. Our structure shows that the native PdxS monomer fashions a single 8α/8β TIM-barrel domain, surrounded by seven additional helices to mediate solvent and interface contacts. A density is present at the active site in the cryoEM map and is interpreted as ribose 5-phosphate. In addition to being the first reconstruction of the PdxS enzyme from a heterogeneous cellular sample, our results reveal a departure from previously published archaeal PdxS crystal structures, lacking the 37 amino acid insertion present in these prior cases. This study demonstrates the potential of applying the cryoID workflow to capture native structural states at atomic resolution for archaeal systems, for which traditional biochemical sample preparation is nontrivial.

Evolutionary Diversity of Dus2 Enzymes Reveals Novel Structural and Functional Features among Members of the RNA Dihydrouridine Synthases Family.

Dihydrouridine (D) is an abundant modified base found in the tRNAs of most living organisms and was recently detected in eukaryotic mRNAs. This base confers significant conformational plasticity to RNA molecules. The dihydrouridine biosynthetic reaction is catalyzed by a large family of flavoenzymes, the dihydrouridine synthases (Dus). So far, only bacterial Dus enzymes and their complexes with tRNAs have been structurally characterized. Understanding the structure-function relationships of eukaryotic Dus proteins has been hampered by the paucity of structural data. Here, we combined extensive phylogenetic analysis with high-precision 3D molecular modeling of more than 30 Dus2 enzymes selected along the tree of life to determine the evolutionary molecular basis of D biosynthesis by these enzymes. Dus2 is the eukaryotic enzyme responsible for the synthesis of D20 in tRNAs and is involved in some human cancers and in the detoxification of β-amyloid peptides in Alzheimer's disease. In addition to the domains forming the canonical structure of all Dus, i.e., the catalytic TIM-barrel domain and the helical domain, both participating in RNA recognition in the bacterial Dus, a majority of Dus2 proteins harbor extensions at both ends. While these are mainly unstructured extensions on the N-terminal side, the C-terminal side extensions can adopt well-defined structures such as helices and beta-sheets or even form additional domains such as zinc finger domains. 3D models of Dus2/tRNA complexes were also generated. This study suggests that eukaryotic Dus2 proteins may have an advantage in tRNA recognition over their bacterial counterparts due to their modularity.

Structural Basis of Salicylic Acid Decarboxylase Reveals a Unique Substrate Recognition Mode and Access Channel.

Salicylic acid (SA) decarboxylase from Trichosporon moniliiforme (TmSdc), which reversibly catalyses the decarboxylation of SA to yield phenol, is of significant interest because of its potential for the production of benzoic acid derivatives under environmentally friendly reaction conditions. TmSdc showed a preference for C2 hydroxybenzoate derivatives, with kcat/Km of SA being 3.2 × 103 M-1 s-1. Here, we presented the first crystal structures of TmSdc, including a complex with SA. The three conserved residues of Glu8, His169, and Asp298 are the catalytic residues within the TIM-barrel domain of TmSdc. Trp239 forms a unique hydrophobic recognition site by interacting with the phenyl ring of SA, while Arg235 is responsible for recognizing the hydroxyl group at the C2 of SA via hydrogen bond interactions. Using a semi-rational combinatorial active-site saturation test, we obtained the TmSdc mutant MT3 (Y64T/P191G/F195V/E302D), which exhibited a 26.4-fold increase in kcat/Km with SA, reaching 8.4 × 104 M-1 s-1. Steered molecular dynamics simulations suggested that the structural changes in MT3 relieved the steric hindrance within the substrate access channel and enlarged the substrate-binding pocket, leading to the increased activity by improving substrate access. Our data elucidate the unique substrate recognition mode and the substrate entrance tunnel of SA decarboxylase.

In Silico Analysis of Fungal and Chloride-Dependent α-Amylases within the Family GH13 with Identification of Possible Secondary Surface-Binding Sites.

This study brings a detailed bioinformatics analysis of fungal and chloride-dependent α-amylases from the family GH13. Overall, 268 α-amylase sequences were retrieved from subfamilies GH13_1 (39 sequences), GH13_5 (35 sequences), GH13_15 (28 sequences), GH13_24 (23 sequences), GH13_32 (140 sequences) and GH13_42 (3 sequences). Eight conserved sequence regions (CSRs) characteristic for the family GH13 were identified in all sequences and respective sequence logos were analysed in an effort to identify unique sequence features of each subfamily. The main emphasis was given on the subfamily GH13_32 since it contains both fungal α-amylases and their bacterial chloride-activated counterparts. In addition to in silico analysis focused on eventual ability to bind the chloride anion, the property typical mainly for animal α-amylases from subfamilies GH13_15 and GH13_24, attention has been paid also to the potential presence of the so-called secondary surface-binding sites (SBSs) identified in complexed crystal structures of some particular α-amylases from the studied subfamilies. As template enzymes with already experimentally determined SBSs, the α-amylases from Aspergillus niger (GH13_1), Bacillus halmapalus, Bacillus paralicheniformis and Halothermothrix orenii (all from GH13_5) and Homo sapiens (saliva; GH13_24) were used. Evolutionary relationships between GH13 fungal and chloride-dependent α-amylases were demonstrated by two evolutionary trees—one based on the alignment of the segment of sequences spanning almost the entire catalytic TIM-barrel domain and the other one based on the alignment of eight extracted CSRs. Although both trees demonstrated similar results in terms of a closer evolutionary relatedness of subfamilies GH13_1 with GH13_42 including in a wider sense also the subfamily GH13_5 as well as for subfamilies GH13_32, GH13_15 and GH13_24, some subtle differences in clustering of particular α-amylases may nevertheless be observed.

Improving the activity and thermostability of GH2 β-glucuronidases via domain reassembly.

Glycoside hydrolase family 2 (GH2) enzymes are generally composed of three domains: TIM-barrel domain (TIM), immunoglobulin-like β-sandwich domain (ISD), and sugar-binding domain (SBD). The combination of these three domains yields multiple structural combinations with different properties. Theoretically, the drawbacks of a given GH2 fold may be circumvented by efficiently reassembling the three domains. However, very few successful cases have been reported. In this study, we used six GH2 β-glucuronidases (GUSs) from bacteria, fungi, or humans as model enzymes and constructed a series of mutants by reassembling the domains from different GUSs. The mutants PGUS-At, GUS-PAA, and GUS-PAP, with reassembled domains from fungal GUSs, showed improved expression levels, activity, and thermostability, respectively. Specifically, compared to the parental enzyme, the mutant PGUS-At displayed 3.8 times higher expression, the mutant GUS-PAA displayed 1.0 timehigher catalytic efficiency (kcat /Km ), and the mutant GUS-PAP displayed 7.5 times higher thermostability at 65°C. Furthermore, two-hybrid mutants, GUS-AEA and GUS-PEP, were constructed with the ISD from a bacterial GUS and SBD and TIM domain from fungal GUSs. GUS-AEA and GUS-PEP showed 30.4% and 23.0% higher thermostability than GUS-PAP, respectively. Finally, molecular dynamics simulations were conducted to uncover the molecular reasons for the increased thermostability of the mutant.

The lid domain is important, but not essential, for catalysis of Escherichia coli pyruvate kinase.

Pyruvate kinase catalyses the final step of the glycolytic pathway in central energy metabolism. The monomeric structure comprises three domains: a catalytic TIM-barrel, a regulatory domain involved in allosteric activation, and a lid domain that encloses the substrates. The lid domain is thought to close over the TIM-barrel domain forming contacts with the substrates to promote catalysis and may be involved in stabilising the activated state when the allosteric activator is bound. However, it remains unknown whether the lid domain is essential for pyruvate kinase catalytic or regulatory function. To address this, we removed the lid domain of Escherichia coli pyruvate kinase type 1 (PKTIM+Reg) using protein engineering. Biochemical analyses demonstrate that, despite the absence of key catalytic residues in the lid domain, PKTIM+Reg retains a low level of catalytic activity and has a reduced binding affinity for the substrate phosphoenolpyruvate. The enzyme retains allosteric activation, but the regulatory profile of the enzyme is changed relative to the wild-type enzyme. Analytical ultracentrifugation and small-angle X-ray scattering data show that, beyond the loss of the lid domain, the PKTIM+Reg structure is not significantly altered and is consistent with the wild-type tetramer that is assembled through interactions at the TIM and regulatory domains. Our results highlight the contribution of the lid domain for facilitating pyruvate kinase catalysis and regulation, which could aid in the development of small molecule inhibitors for pyruvate kinase and related lid-regulated enzymes.

Structural Insights into the Molecular Evolution of the Archaeal Exo-β-d-Glucosaminidase.

The archaeal exo-β-d-glucosaminidase (GlmA), a thermostable enzyme belonging to the glycosidase hydrolase (GH) 35 family, hydrolyzes chitosan oligosaccharides into monomer glucosamines. GlmA is a novel enzyme in terms of its primary structure, as it is homologous to both GH35 and GH42 β-galactosidases. The catalytic mechanism of GlmA is not known. Here, we summarize the recent reports on the crystallographic analysis of GlmA. GlmA is a homodimer, with each subunit comprising three distinct domains: a catalytic TIM-barrel domain, an α/β domain, and a β1 domain. Surprisingly, the structure of GlmA presents features common to GH35 and GH42 β-galactosidases, with the domain organization resembling that of GH42 β-galactosidases and the active-site architecture resembling that of GH35 β-galactosidases. Additionally, the GlmA structure also provides critical information about its catalytic mechanism, in particular, on how the enzyme can recognize glucosamine. Finally, we postulate an evolutionary pathway based on the structure of an ancestor GlmA to extant GH35 and GH42 β-galactosidases.

Enhancing the thermostability of β-glucuronidase from T. pinophilus enables the biotransformation of glycyrrhizin at elevated temperature

Glycosidases have been widely used in natural products modification by precisely hydrolyzing sugar moieties to modulate the biological activities. However, the low stability of the glycosidases under harsh conditions and the low solubility of substrate have largely impeded the biotransformation efficiency. To solve this problem, in this study, we selected the biotransformation of glycyrrhizin (GL) into glycyrrhetinic acid 3-O-mono-β-D-glucuronide (GAMG) catalyzed by β-glucuronidase from T. pinophilus Li-93 (TpGUS79A-P) as a model reaction. We combined three strategies including random mutagenesis, computation-aided design and rational introduction of “clamp” to TIM-barrel domain to enhance the thermostability of TpGUS79A-P, and one mutant TpGUS79A-P4 was obtained with thermostability at 55 °C increased by 2.9-fold. The enzymatic properties of TpGUS79A-P4 were extensively characterized. Finally, a novel high-temperature biotransformation process of GL into GAMG catalyzed by TpGUS79A-P4 was established at 50 °C, with GAMG concentration increased by 84% than that at 30 °C.

Alternative conformation induced by substrate binding for Arabidopsis thalianaN6-methyl-AMP deaminase.

Adenosine deaminase is involved in adenosine degradation and salvage pathway, and plays important physiological roles in purine metabolism. Recently, a novel type of adenosine deaminase-like protein has been identified, which displays deamination activity toward N6-methyl-adenosine monophosphate but not adenosine or AMP, and was consequently named N6-methyl-AMP deaminase (MAPDA). The underlying structural basis of MAPDA recognition and catalysis is poorly understood. Here, we present the crystal structures of MAPDA from Arabidopsis thaliana in the free and in the ligand-bound forms. The protein contains a conserved (β/α)8 Tim-barrel domain and a typical zinc-binding site, but it also exhibits idiosyncratic local differences for two flexible helices important for substrate binding. The extensive interactions between the N6-methyl-AMP substrate or the inosine monophosphate product and the enzyme were identified, and subsequently evaluated by the deamination activity assays. Importantly, each structure reported here represents a different stage of the catalytic pathway and their structural differences suggested that the enzyme can exist in two distinct conformational states. The open state switches to the closed one upon the binding of ligands, brought about by the two critical helices. Our structural studies provide the first look of this important metabolic enzyme and shed lights on its catalytic pathway, which holds promise for the structure-based drug design for MAPDA-related diseases.

Crystal structure and substrate recognition mechanism of Aspergillus oryzae isoprimeverose-producing enzyme

Isoprimeverose-producing enzymes (IPases) release isoprimeverose (α-d-xylopyranosyl-(1 → 6)-d-glucopyranose) from the non-reducing end of xyloglucan oligosaccharides. Aspergillus oryzae IPase (IpeA) is classified as a member of the glycoside hydrolase family 3 (GH3); however, it has unusual substrate specificity compared with other GH3 enzymes. Xylopyranosyl branching at the non-reducing ends of xyloglucan oligosaccharides is vital for IpeA activity. We solved the crystal structure of IpeA with isoprimeverose at 2.4 Å resolution, showing that the structure of IpeA formed a dimer and was composed of three domains: an N-terminal (β/α)8 TIM-barrel domain, α/β/α sandwich fold domain, and a C-terminal fibronectin-like domain. The catalytic TIM-barrel domain possessed a catalytic nucleophile (Asp300) and acid/base (Glu524) residues. Interestingly, we found that the cavity of the active site of IpeA was larger than that of other GH3 enzymes, and subsite −1′ played an important role in its activity. The glucopyranosyl and xylopyranosyl residues of isoprimeverose were located at subsites −1 and −1′, respectively. Gln58 and Tyr89 contributed to the interaction with the xylopyranosyl residue of isoprimeverose through hydrogen bonding and stacking effects, respectively. Our findings provide new insights into the substrate recognition of GH3 enzymes.

Steric hindrance controls pyridine nucleotide specificity of a flavin-dependent NADH:quinone oxidoreductase.

The crystal structure of the NADH:quinone oxidoreductase PA1024 has been solved in complex with NAD+ to 2.2 Å resolution. The nicotinamide C4 is 3.6 Å from the FMN N5 atom, with a suitable orientation for facile hydride transfer. NAD+ binds in a folded conformation at the interface of the TIM-barrel domain and the extended domain of the enzyme. Comparison of the enzyme-NAD+ structure with that of the ligand-free enzyme revealed a different conformation of a short loop (75-86) that is part of the NAD+ -binding pocket. P78, P82, and P84 provide internal rigidity to the loop, whereas Q80 serves as an active site latch that secures the NAD+ within the binding pocket. An interrupted helix consisting of two α-helices connected by a small three-residue loop binds the pyrophosphate moiety of NAD+ . The adenine moiety of NAD+ appears to π-π stack with Y261. Steric constraints between the adenosine ribose of NAD+ , P78, and Q80, control the strict specificity of the enzyme for NADH. Charged residues do not play a role in the specificity of PA1024 for the NADH substrate.

LKB1 regulates PRMT5 activity in breast cancer.

Protein arginine methyltransferase 5 (PRMT5) is the main enzyme responsible for the symmetrical dimethylation of arginine residues on target proteins in both the cytoplasm and the nucleus. Though its activity has been associated with tumor progression in various cancers, the expression pattern of this oncoprotein has been scarcely studied in breast cancer. In the current work, we analyzed its expression in a large cohort of breast cancer patients, revealing higher nuclear PRMT5 levels in ERα-positive tumors and an association with prolonged disease free and overall survival. Interestingly, high PRMT5 nuclear expression was also associated with higher nuclear liver kinase B1 (LKB1), suggesting that a functional relationship may occur. Consistently, several approaches provided evidence that PRMT5 and LKB1 interact directly in the cytoplasm of mammary epithelial cells. Moreover, although PRMT5 is not able to methylate LKB1, we found that PRMT5 is a bona fade substrate for LKB1. We identified T132, 139 and 144 residues, located in the TIM-Barrel domain of PRMT5, as target sites for LKB1 phosphorylation. The point mutation of PRMT5 T139/144 to A139/144 drastically decreased its methyltransferase activity, due probably to the loss of its interaction with regulatory proteins such as MEP50, pICln and RiOK1. In addition, modulation of LKB1 expression modified PRMT5 activity, highlighting a new regulatory mechanism that could have clinical implications.

Hidden Sequence Repeats: Additional Evidence for the Origin of TIM-Barrel Family

Most proteins adopt an approximate structural symmetry. However, they have no symmetry detectable in their sequences and it is unclear for most of these proteins whether their structural symmetry originates from duplication. As one of the six popular folds (super-folds) possessing an approximate structural symmetry, the triosephosphate isomerase barrel (TIM-barrel) domain has been widely studied. Using modified recurrent quantification analysis of primary sequences, we identified the same 2-, 3-, and 4-fold symmetry pattern as their tertiary structures. This result indicates that the symmetry in tertiary structure is coded by symmetry in the primary sequence and that the TIM-barrel adopts a 2-, 3-, or 4-fold repeat pattern during evolution. This discovery will be useful for understanding the evolutionary mechanisms of this protein family and the symmetry pattern that may be a clue into the ancient origin of duplication of half-barrels or the β a unit.

Purification, crystallization and structural elucidation of D-galactaro-1,4-lactone cycloisomerase from Agrobacterium tumefaciens involved in pectin degradation.

Pectin is found in the cell wall of plants and is often discarded as waste. A number of research groups are interested in redirecting this biomass waste stream for the production of fuel and bulk chemicals. The primary monomeric subunit of this polysaccharide is D-galacturonate, a six-carbon acid sugar that is degraded in a five-step pathway to central metabolic intermediates by some bacteria, including Agrobacterium tumefaciens. In the third step of the pathway, D-galactaro-1,4-lactone is converted to 2-keto-3-deoxy-L-threo-hexarate by a member of the mandelate racemase subgroup of the enolase superfamily with a novel activity for the superfamily. The 1.6 Å resolution structure of this enzyme was determined, revealing an overall modified (β/α)7β TIM-barrel domain, a hallmark of the superfamily. D-Galactaro-1,4-lactone was manually docked into the active site located at the interface between the N-terminal lid domain and the C-terminal barrel domain. On the basis of the position of the lactone in the active site, Lys166 is predicted to be the active-site base responsible for abstraction of the α proton. His296 on the opposite side of the active site is predicted to be the general acid that donates a proton to the β carbon as the lactone ring opens. The lactone ring appears to be oriented within the active site by stacking interactions with Trp298.

Structure-function relationships in Gan42B, an intracellular GH42 β-galactosidase from Geobacillus stearothermophilus.

Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic soil bacterium that contains a battery of degrading enzymes for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. A 9.4 kb gene cluster has recently been characterized in G. stearothermophilus that encodes a number of galactan-utilization elements. A key enzyme of this degradation system is Gan42B, an intracellular GH42 β-galactosidase capable of hydrolyzing short β-1,4-galactosaccharides into galactose units, making it of high potential for various biotechnological applications. The Gan42B monomer is made up of 686 amino acids, and based on sequence homology it was suggested that Glu323 is the catalytic nucleophile and Glu159 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Gan42B (at 2.45 Å resolution) and its catalytic mutant E323A (at 2.50 Å resolution), as determined by X-ray crystallography, are reported. These structures demonstrate that the three-dimensional structure of the Gan42B monomer generally correlates with the overall fold observed for GH42 proteins, consisting of three main domains: an N-terminal TIM-barrel domain, a smaller mixed α/β domain, and the smallest all-β domain at the C-terminus. The two catalytic residues are located in the TIM-barrel domain in a pocket-like active site such that their carboxylic functional groups are about 5.3 Å from each other, consistent with a retaining mechanism. The crystal structure demonstrates that Gan42B is a homotrimer, resembling a flowerpot in general shape, in which each monomer interacts with the other two to form a cone-shaped tunnel cavity in the centre. The cavity is ∼35 Å at the wide opening and ∼5 Å at the small opening and ∼40 Å in length. The active sites are situated at the interfaces between the monomers, so that every two neighbouring monomers participate in the formation of each of the three active sites of the trimer. They are located near the small opening of the cone tunnel, all facing the centre of the cavity. The biological relevance of this trimeric structure is supported by independent results obtained from gel-permeation chromatography. These data and their comparison to the structural data of related GH42 enzymes are used for a more general discussion concerning structure-activity aspects in this GH family.

The stability of the TIM-barrel domain of a psychrophilic chitinase

Chitinase 60 from the psychrophilic bacterium Moritella marina (MmChi60) is a four-domain protein whose structure revealed flexible hinge regions between the domains, yielding conformations in solution that range from fully extended to compact. The catalytic domain is a shallow-grooved TIM-barrel. Heat-induced denaturation experiments of the wild-type and mutants resulting from the deletions of the two-Ig-like domains and the chitin binding domain reveal calorimetric profiles that are consistent with non-collaborative thermal unfolding of the individual domains, a property that must be associated to the “hinge-regions”. The calorimetric measurements of the (β/α)8 catalytic domain reveal that the thermal unfolding is a slow-relaxation transition exhibiting a stable, partially structured intermediate state. Circular dichroism provides evidence that the intermediate exhibits features of a molten globule i.e., loss of tertiary structure while maintaining the secondary structural elements of the native. GdnHCl-induced denaturation studies of the TIM-barrel demonstrate an extraordinarily high resistance to the denaturant. Slow-relaxation kinetics characterize the unfolding with equilibration times exceeding six days, a property that is for the first time observed for a psychrophilic TIM barrel. On the other hand, the thermodynamic stability is ΔG=6.75±1.3kcal/mol, considerably lower than for structural-insertions-containing barrels. The mutant E153Q used for the crystallographic studies of MmChi60 complexes with NAG ligands has a much lower stability than the wild-type.

Structure-specificity relationships in Abp, a GH27 β-L-arabinopyranosidase from Geobacillus stearothermophilus T6.

L-Arabinose sugar residues are relatively abundant in plants and are found mainly in arabinan polysaccharides and in other arabinose-containing polysaccharides such as arabinoxylans and pectic arabinogalactans. The majority of the arabinose units in plants are present in the furanose form and only a small fraction of them are present in the pyranose form. The L-arabinan-utilization system in Geobacillus stearothermophilus T6, a Gram-positive thermophilic soil bacterium, has recently been characterized, and one of the key enzymes was found to be an intracellular β-L-arabinopyranosidase (Abp). Abp, a GH27 enzyme, was shown to remove β-L-arabinopyranose residues from synthetic substrates and from the native substrates sugar beet arabinan and larch arabinogalactan. The Abp monomer is made up of 448 amino acids, and based on sequence homology it was suggested that Asp197 is the catalytic nucleophile and Asp255 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Abp (at 2.28 Å resolution) and its catalytic mutant Abp-D197A with (at 2.20 Å resolution) and without (at 2.30 Å resolution) a bound L-arabinose product are reported as determined by X-ray crystallography. These structures demonstrate that the three-dimensional structure of the Abp monomer correlates with the general fold observed for GH27 proteins, consisting of two main domains: an N-terminal TIM-barrel domain and a C-terminal all-β domain. The two catalytic residues are located in the TIM-barrel domain, such that their carboxylic functional groups are about 5.9 Å from each other, consistent with a retaining mechanism. An isoleucine residue (Ile67) located at a key position in the active site is shown to play a critical role in the substrate specificity of Abp, providing a structural basis for the high preference of the enzyme towards arabinopyranoside over galactopyranoside substrates. The crystal structure demonstrates that Abp is a tetramer made up of two `open-pincers' dimers, which clamp around each other to form a central cavity. The four active sites of the Abp tetramer are situated on the inner surface of this cavity, all opening into the central space of the cavity. The biological relevance of this tetrameric structure is supported by independent results obtained from size-exclusion chromatography (SEC), dynamic light-scattering (DLS) and small-angle X-ray scattering (SAXS) experiments. These data and their comparison to the structural data of related GH27 enzymes are used for a more general discussion concerning structure-selectivity aspects in this glycoside hydrolase (GH) family.

Structures of bacterial biosynthetic arginine decarboxylases

Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0 Å resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1 Å resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a β-sandwich domain, as well as two smaller helical domains. The TIM-barrel andβ-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the β-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, π-stacking and van der Waals interactions.

A glycyl free radical as the precursor in the synthesis of carbon monoxide and cyanide by the [FeFe]-hydrogenase maturase HydG

HydG uses tyrosine to synthesize the CN −/CO ligands of [FeFe]-hydrogenase active site. We have mutated two of the [4Fe–4S]-cluster cysteine ligands of the HydG C-terminal domain (CTD) to serine. The double mutant can still synthesize CN − but not CO. In a mutant lacking the CTD both CN − and CO synthesis are abolished. Like in ThiH, the initial steps of CN − synthesis are carried out in the TIM-barrel domain of HydG but some component(s) of the CTD are later needed. The mutants indicate that CO synthesis is metal-based and occurs in the CTD. We postulate that CN −/CO synthesis is initiated by H 2N– CH– CO 2 - . Fragmentation of this radical into H 2N– CH 2 and CO 2 or H 2C NH and CO 2 - provides plausible precursors for CN −/CO synthesis.