Crystal structure of a family 4 uracil-DNA glycosylase from Thermus thermophilus HB8

Uracil-DNA glycosylase (UDG; EC 3.2.2.-) removes uracil from DNA to initiate DNA base excision repair. Since hydrolytic deamination of cytosine to uracil is one of the most frequent DNA-damaging events in all cells, UDG is an essential enzyme for maintaining the integrity of genomic information. For the first time, we report the crystal structure of a family 4 UDG from Thermus thermophilus HB8 (TthUDG) complexed with uracil, solved at 1.5 angstroms resolution. As opposed to UDG enzymes in its other families, TthUDG possesses a [4Fe-4S] cluster. This iron-sulfur cluster, which is distant from the active site, interacts with loop structures and has been suggested to be unessential to the activity but necessary for stabilizing the loop structures. In addition to the iron-sulfur cluster, salt-bridges and ion pairs on the molecular surface and the presence of proline on loops and turns is thought to contribute to the enzyme's thermostability. Despite very low levels of sequence identity with Escherichia coli and human UDGs (family 1) and E.coli G:T/U mismatch-specific DNA glycosylase (MUG) (family 2), the topology and order of secondary structures of TthUDG are similar to those of these distant relatives. Furthermore, the coordinates of the core structure formed by beta-strands are almost the same. Positive charge is distributed over the active-site groove, where TthUDG would bind DNA strands, as do UDG enzymes in other families. TthUDG recognizes uracil specifically in the same manner as does human UDG (family 1), rather than guanine in the complementary strand DNA, as does E.coli MUG (family 2). These results suggest that the mechanism by which family 4 UDGs remove uracils from DNA is similar to that of family 1 enzymes.     

Functional changes in a novel uracil-DNA glycosylase determined by mutational analyses

Uracil-DNA glycosylase (UDG) is a ubiquitous enzyme found in bacteria and eukaryotes, which removes uracil residues from DNA strands. Methanococcus jannaschii UDG (MjUDG), a novel monofunctional glycosylase, contains a helix-hairpin-helix (HhH) motif and a Gly/Pro rich loop (GPD region), which is important for catalytic activity; it shares these features with other glycosylases, such as endonuclease III. First, to examine the role of two conserved amino acid residues (Asp150 and Tyr152) in the HhH-GPD region of MjUDG, mutant MjUDG proteins were constructed, in which Asp150 was replaced with either Glu or Trp (D150E and D150W), and Tyr152 was replaced with either Glu or Asn (Y152E and Y152N). Mutant D150W completely lacked DNA glycosylase activity, whereas D150E displayed reduced activity of about 70% of the wild type value. However, the mutants Y152E and Y152N retained unchanged levels of UDG activity. We also replaced Glu132 in the HhH motif with a lysine residue equivalent to Lys120 in endonuclease III. This mutation converted the enzyme into a bifunctional glycosylase/AP lyase capable of both removing uracil at a glycosylic bond and cleaving the phosphodiester backbone at an AP site. Mutant E132K catalyzes a β-elimination reaction at the AP site via uracil excision and forms a Schiff base intermediate in the form of a protein-DNA complex. This text was submitted by the authors in English.     

Insights from xanthine and uracil DNA glycosylase activities of bacterial and human SMUG1: switching SMUG1 to UDG

Single-strand-selective monofunctional uracil DNA glycosylase (SMUG1) belongs to Family 3 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that a bacterial SMUG1 ortholog in Geobacter metallireducens (Gme) and the human SMUG1 enzyme are not only UDGs but also xanthine DNA glycosylases (XDGs). In addition, mutational analysis and molecular dynamics (MD) simulations of Gme SMUG1 identify important structural determinants in conserved motifs 1 and 2 for XDG and UDG activities. Mutations at M57 (M57L) and H210 (H210G, H210M, and H210N), both of which are involved in interactions with the C2 carbonyl oxygen in uracil or xanthine, cause substantial reductions in XDG and UDG activities. Increased selectivity is achieved in the A214R mutant of Gme SMUG1, which corresponds to a position involved in base flipping. This mutation results in an activity profile resembling a human SMUG1-like enzyme as exemplified by the retention of UDG activity on mismatched base pairs and weak XDG activity. MD simulations indicate that M57L increases the flexibility of the motif 2 loop region and specifically A214, which may account for the reduced catalytic activity. G60Y completely abolishes XDG and UDG activity, which is consistent with a modeled structure in which G60Y blocks the entry of either xanthine or uracil to the base binding pocket. Most interestingly, a proline substitution at the G63 position switches the Gme SMUG1 enzyme to an exclusive UDG as demonstrated by the uniform excision of uracil in both double-stranded and single-stranded DNA and the complete loss of XDG activity. MD simulations indicate that a combination of a reduced free volume and altered flexibility in the active-site loops may underlie the dramatic effects of the G63P mutation on the activity profile of SMUG1. This study offers insights on the important role that modulation of conformational flexibility may play in defining specificity and catalytic efficiency.     

Crystal structure of cytochrome P450 MoxA from Nonomuraea recticatena (CYP105)

Cytochrome P450 MoxA (P450moxA) from a rare actinomycete Nonomuraea recticatena belongs to the CYP105 family and exhibits remarkably broad substrate specificity. Here, we demonstrate that P450moxA acts on several luciferin derivatives, which were originally identified as substrates of the human microsomal P450s. We also describe the crystal structure of P450moxA in substrate-free form. Structural comparison with various bacterial and human microsomal P450s reveals that the P450moxA structure is most closely related to that of the fungal nitric oxide reductase P450nor (CYP55A1). Final refined model of P450moxA comprises almost all the residues, including the "BC-loop" and "FG-loop" regions pivotal for substrate recognition, and the current structure thus defines a well-ordered substrate-binding pocket. Clear electron density map reveals that the MES molecule is bound to the substrate-binding site, and the sixth coordination position of the heme iron is not occupied by a water molecule, probably due to the presence of MES molecule in the vicinity of the heme. The unexpected binding of the MES molecule might reflect the ability of P450moxA to accommodate a broad range of structurally diverse compounds.     

Crystal structure of a novel polyisoprenoid-binding protein from Thermus thermophilus HB8

The isoprenoid quinones exist widely among prokaryotes and eukaryotes. They play essential roles in respiratory electron transport and in controlling oxidative stress and gene regulation. In the isoprenoid quinone biosynthetic pathway, polyprenyl pyrophosphates are used as isoprenoid side-chain precursors. Here we report the crystal structure of a novel polyprenyl pyrophosphate binding protein, TT1927b, from Thermus thermophilus HB8, complexed with its ligand. This protein belongs to the YceI-like family in the Pfam database, and its sequence homologs are present in a broad range of bacteria and archaea. The structure consists of an extended, eight-stranded, antiparallel beta-barrel. In the hydrophobic pore of the barrel, the protein binds the polyisoprenoid chain by hydrophobic interactions. Its overall structure resembles the lipocalin fold, but there is no sequence homology between TT1927b and the lipocalin family of proteins.     

Asp302 determines potassium dependence of a RadA recombinase from Methanococcus voltae

Archaeal RadA/Rad51 are close homologues of eukaryal Rad51/DMC1. Such recombinases, as well as their bacterial RecA orthologues, form helical nucleoprotein filaments in which a hallmark strand exchange reaction occurs between homologous DNA substrates. Our recent ATPase and structure studies on RadA recombinase from Methanococcus voltae have suggested that not only magnesium but also potassium ions are absorbed at the ATPase center. Potassium, but not sodium, stimulates the ATP hydrolysis reaction with an apparent dissociation constant of approximately 40 mM. The minimal inhibitory effect by 40 mM NaCl further suggests that the protein does not have adequate affinity for sodium. The wild-type protein's strand exchange activity is also stimulated by potassium with an apparent dissociation constant of approximately 35 mM. We made site-directed mutations at the potassium-contacting residues Glu151 and Asp302. The mutant proteins are expectedly defective in promoting ATP hydrolysis. Similar potassium preference in strand exchange is observed for the E151D and E151K proteins. The D302K protein, however, shows comparable strand exchange efficiencies in the presence of either potassium or sodium. Crystallized E151D filaments reveal a potassium-dependent conformational change similar to what has previously been observed with the wild-type protein. We interpret these data as suggesting that both ATP hydrolysis and DNA strand exchange requires accessibility to an "active" conformation similar to the crystallized ATPase-active form in the presence of ATP, Mg2+ and K+.     

Molecular mechanism of ADP-ribose hydrolysis by human NUDT5 from structural and kinetic studies

Human NUDT5 (hNUDT5) is an ADP-ribose (ADPR) pyrophosphatase (ADPRase) that plays important roles in controlling the intracellular levels of ADPR and preventing non-enzymatic ADP-ribosylation of proteins by hydrolyzing ADPR to AMP and ribose 5′-phosphate. We report the crystal structure of hNUDT5 in complex with a non-hydrolyzable ADPR analogue, α,β-methyleneadenosine diphosphoribose, and three Mg^2 + ions representing the transition state of the enzyme during catalysis. Analysis of this structure and comparison with previously reported hNUDT5 structures identify key residues involved in substrate binding and catalysis. In the transition-state structure, three metal ions are bound at the active site and are coordinated by surrounding residues and water molecules. A conserved water molecule is at an ideal position for nucleophilic attack on the α-phosphate of ADPR. The side chain of Glu166 on loop L9 changes its conformation to interact with the conserved water molecule compared with that in the substrate-bound structure and appears to function as a catalytic base. Mutagenesis and kinetic studies show that Trp28 and Trp46 are important for the substrate binding; Arg51 is involved in both the substrate binding and the catalysis; and Glu112 and Glu116 of the Nudix motif, Glu166 on loop L9, and Arg111 are critical for the catalysis. The structural and biochemical data together reveal the molecular basis of the catalytic mechanism of ADPR hydrolysis by hNUDT5. Specifically, Glu166 functions as a catalytic base to deprotonate a conserved water molecule that acts as a nucleophile to attack the α-phosphate of ADPR, and three Mg^2 + ions are involved in the activation of the nucleophile and the binding of the substrate. Structural comparison of different ADPRases also suggests that most dimeric ADPRases may share a similar catalytic mechanism of ADPR hydrolysis.     

Biochemical properties of single-stranded DNA-binding protein from Mycobacterium smegmatis,a fast-growing mycobacterium and its physical and functional interaction with uracil DNA glycosylases

The single-stranded DNA-binding proteins (SSBs) are vital to virtually all DNA functions. Here, we report on the biochemical properties of SSB from a fast-growing mycobacteria, Mycobacterium smegmatis, and the interaction of the homotetrameric SSBs with uracil DNA glycosylases (UDGs) from M. smegmatis (Msm), Mycobacterium tuberculosis (Mtu) and Escherichia coli (Eco). UDG is a crucial DNA repair enzyme, which removes the promutagenic uracil residues. MsmSSB stimulates activity of the homologous Msm UDG and of the heterologous Mtu-, and Eco-UDGs. On the contrary, while the MtuSSB stimulates the Mtu UDG, it inhibits the other two UDGs. Although the MsmSSB shares 84% identity with MtuSSB, the two are strikingly different, in that MsmSSB contains a glycine-rich segment (11 out of 13 residues) in the spacer connecting the N-terminal DNA-binding domain with the C-terminal acidic tail. While the DNA-binding properties of MsmSSB, such as its affinity to oligomeric DNA, requirement of minimum size DNA and the modes of interaction are indistinguishable from those of Eco-, and Mtu-SSBs, it is unclear if the glycine-rich segment confers structural advantage to MsmSSB, responsible for its stimulatory effect on all UDGs tested. More importantly, by using a small polypeptide inhibitor of UDGs, and the deletion mutants of SSBs, we suggest that the C-terminal acidic tail of the SSBs interacts within the DNA-binding groove of the UDGs, and propose a role for SSBs in the recruitment of UDGs to the damaged DNA.     

Crystal structure of Thermotoga maritima 4-alpha-glucanotransferase and its acarbose complex: implications for substrate specificity and catalysis

4-alpha-Glucanotransferase (GTase) is an essential enzyme in alpha-1,4-glucan metabolism in bacteria and plants. It catalyses the transfer of maltooligosaccharides from an 1,4-alpha-D-glucan molecule to the 4-hydroxyl group of an acceptor sugar molecule. The crystal structures of Thermotoga maritima GTase and its complex with the inhibitor acarbose have been determined at 2.6A and 2.5A resolution, respectively. The GTase structure consists of three domains, an N-terminal domain with the (beta/alpha)(8) barrel topology (domain A), a 65 residue domain, domain B, inserted between strand beta3 and helix alpha6 of the barrel, and a C-terminal domain, domain C, which forms an antiparallel beta-structure. Analysis of the complex of GTase with acarbose has revealed the locations of five sugar-binding subsites (-2 to +3) in the active-site cleft lying between domain B and the C-terminal end of the (beta/alpha)(8) barrel. The structure of GTase closely resembles the family 13 glycoside hydrolases and conservation of key catalytic residues previously identified for this family is consistent with a double-displacement catalytic mechanism for this enzyme. A distinguishing feature of GTase is a pair of tryptophan residues, W131 and W218, which, upon the carbohydrate inhibitor binding, form a remarkable aromatic "clamp" that captures the sugar rings at the acceptor-binding sites +1 and +2. Analysis of the structure of the complex shows that sugar residues occupying subsites from -2 to +2 engage in extensive interactions with the protein, whereas the +3 glucosyl residue makes relatively few contacts with the enzyme. Thus, the structure suggests that four subsites, from -2 to +2, play the dominant role in enzyme-substrate recognition, consistent with the observation that the smallest donor for T.maritima GTase is maltotetraose, the smallest chain transferred is a maltosyl unit and that the smallest residual fragment after transfer is maltose. A close similarity between the structures of GTase and oligo-1,6-glucosidase has allowed the structural features that determine differences in substrate specificity of these two enzymes to be analysed.     

Crystal Structure Analysis of DNA Uridine Endonuclease Mth212 Bound to DNA

The reliable repair of pre-mutagenic U/G mismatches that originated from hydrolytic cytosine deamination is crucial for the maintenance of the correct genomic information. In most organisms, any uracil base in DNA is attacked by uracil DNA glycosylases (UDGs), but at least in Methanothermobacter thermautotrophicus ΔH, an alternative strategy has evolved. The exonuclease III homologue Mth212 from the thermophilic archaeon M. thermautotrophicus ΔH exhibits a DNA uridine endonuclease activity in addition to the apyrimidinic/apurinic site endonuclease and 3′ → 5′exonuclease functions. Mth212 alone compensates for the lack of a UDG in a single-step reaction thus substituting the two-step pathway that requires the consecutive action of UDG and apyrimidinic/apurinic site endonuclease.In order to gain deeper insight into the structural basis required for the specific uridine recognition by Mth212, we have characterized the enzyme by means of X-ray crystallography. Structures of Mth212 wild-type or mutant proteins either alone or in complex with DNA substrates and products have been determined to a resolution of up to 1.2 Å, suggesting key residues for the uridine endonuclease activity. The insertion of the side chain of Arg209 into the DNA helical base stack resembles interactions observed in human UDG and seems to be crucial for the uridine recognition. In addition, Ser171, Asn153, and Lys125 in the substrate binding pocket appear to have important functions in the discrimination of aberrant uridine against naturally occurring thymidine and cytosine residues in double-stranded DNA.     

Uracil‐DNA glycosylases—Structural and functional perspectives on an essential family of DNA repair enzymes

Uracil‐DNA glycosylases (UDGs) are evolutionarily conserved DNA repair enzymes that initiate the base excision repair pathway and remove uracil from DNA. The UDG superfamily is classified into six families based on their substrate specificity. This review focuses on the family I enzymes since these are the most extensively studied members of the superfamily. The structural basis for substrate specificity and base recognition as well as for DNA binding, nucleotide flipping and catalytic mechanism is discussed in detail. Other topics include the mechanism of lesion search and molecular mimicry through interaction with uracil‐DNA glycosylase inhibitors. The latest studies and findings detailing structure and function in the UDG superfamily are presented.     

Single-stranded DNA binding protein facilitates specific enrichment of circular DNA molecules using rolling circle amplification

Many techniques in molecular biology require the use of pure nucleic acids in general and circular DNA (plasmid or mitochondrial) in particular. We have developed a method to separate these circular molecules from a mixture containing different species of nucleic acids using rolling circle amplification (RCA). RCA of plasmid or genomic DNA using random hexamers and bacteriophage Phi29 DNA polymerase has become increasingly popular for the amplification of template DNA in DNA sequencing protocols. Recently, we reported that the mutant single-stranded DNA binding protein (SSB) from Thermus thermophilus (TthSSB) HB8 eliminates nonspecific DNA products in RCA reactions. We developed this method for separating circular nucleic acids from a mixture having different species of nucleic acids. Use of the mutant TthSSB resulted in an enhancement of plasmid or mitochondrial DNA content in the amplified product by approximately 500×. The use of mutant TthSSB not only promoted the amplification of circular target DNA over the background but also could be used to enhance the amplification of circular targets over linear targets.     

Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: structure and glycosylase mechanism revisited

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil residues from single-stranded or duplex DNA, producing free uracil and abasic DNA. Here we report the high-resolution crystal structures of free UDG from Escherichia coli strain B (1.60 A), its complex with uracil (1.50 A), and a second active-site complex with glycerol (1.43 A). These represent the first high-resolution structures of a prokaryotic UDG to be reported. The overall structure of the E. coli enzyme is more similar to the human UDG than the herpes virus enzyme. Significant differences between the bacterial and viral structures are seen in the side-chain positions of the putative general-acid (His187) and base (Asp64), similar to differences previously observed between the viral and human enzymes. In general, the active-site loop that contains His187 appears preorganized in comparison with the viral and human enzymes, requiring smaller substrate-induced conformational changes to bring active-site groups into catalytic position. These structural differences may be related to the large differences in the mechanism of uracil recognition used by the E. coli and viral enzymes. The pH dependence of k(cat) for wild-type UDG and the D64N and H187Q mutant enzymes is consistent with general-base catalysis by Asp64, but provides no evidence for a general-acid catalyst. The catalytic mechanism of UDG is critically discussed with respect to these results.     

The crystal structure of l-sorbose reductase from Gluconobacter frateurii complexed with NADPH and l-sorbose

l-Sorbose reductase from Gluconobacter frateurii (SR) is an NADPH-dependent oxidoreductase. SR preferentially catalyzes the reversible reaction between d-sorbitol and l-sorbose with high substrate specificity. To elucidate the structural basis of the catalytic mechanism and the substrate specificity of SR, we have determined the structures of apo-SR, SR in complex with NADPH, and the inactive mutant (His116Leu) of SR in complex with NADPH and l-sorbose at 2.83 Å, 1.90 Å, and 1.80 Å resolutions, respectively. Our results show that SR belongs to the short-chain dehydrogenase/reductase (SDR) family and forms a tetrameric structure. Although His116 is not conserved among SDR family enzymes, the structures of SR have revealed that His116 is important for the stabilization of the proton relay system and for active-site conformation as a fourth catalytic residue. In the ternary complex structure, l-sorbose is recognized by 11 hydrogen bonds. Site-directed mutagenesis of residues around the l-sorbose-binding site has shown that the loss of almost full enzymatic activity was caused by not only the substitution of putative catalytic residues but also the substitution of the residue used for the recognition of the C4 hydroxyl groups of l-sorbose (Glu154) and of the residues used for the construction of the substrate-binding pocket (Cys146 and Gly188). The recognition of the C4 hydroxyl group of l-sorbose would be indispensable for the substrate specificity of SR, which recognizes only l-sorbose and d-sorbitol but not other sugars. Our results indicated that these residues were crucial for the substrate recognition and specificity of SR.     

Functional Importance of Bacteriophage ?29 DNA Polymerase Residue Tyr148 in Primer-terminus Stabilisation at the 3′-5′ Exonuclease Active Site

Recent crystallographic resolution of ?29 DNA polymerase complexes with ssDNA at its 3′-5′ exonuclease active site has allowed the identification of residues Pro129 and Tyr148 as putative ssDNA ligands, the latter being conserved in the Kx₂h motif of proofreading family B DNA polymerases. Single substitution of ?29 DNA polymerase residue Tyr148 to Ala rendered an enzyme with a reduced capacity to stabilize the binding of the primer terminus at the 3′-5′ exonuclease active site, not having a direct role in the catalysis of the reaction. Analysis of the 3′-5′ exonuclease on primer/template structures showed a critical role for residue Tyr148 in the proofreading of DNA polymerisation errors. In addition, Tyr148 is not involved in coupling polymerisation to strand displacement in contrast to the catalytic residues responsible for the exonuclease reaction, its role being restricted to stabilisation of the frayed 3′ terminus at the exonuclease active site. Altogether, the results lead us to extend the consensus sequence of the above motif of proofreading family B DNA polymerases into Kx₂hxA. The different solutions adopted by proofreading DNA polymerases to stack the 3′ terminus at the exonuclease site are discussed. In addition, the results obtained with mutants at ?29 DNA polymerase residue Pro129 allow us to rule out a functional role as ssDNA ligand for this residue.     

Binding of specific DNA base-pair mismatches by N-methylpurine-DNA glycosylase and its implication in initial damage recognition

Most DNA glycosylases including N-methylpurine-DNA glycosylase (MPG), which initiate DNA base excision repair, have a wide substrate range of damaged or altered bases in duplex DNA. In contrast, uracil-DNA glycosylase (UDG) is specific for uracil and excises it from both single-stranded and duplex DNAs. Here we show by DNA footprinting analysis that MPG, but not UDG, bound to base-pair mismatches especially to less stable pyrimidine-pyrimidine pairs, without catalyzing detectable base cleavage. Thermal denaturation studies of these normal and damaged (e.g. 1,N(6)-ethenoadenine, varepsilonA) base mispairs indicate that duplex instability rather than exact fit of the flipped out damaged base in the catalytic pocket is a major determinant in the initial recognition of damage by MPG. Finally, based on our determination of binding affinity and catalytic efficiency we conclude that the initial recognition of substrate base lesions by MPG is dependent on the ease of flipping of the base from unstable pairs to a flexible catalytic pocket.     

In silico evidence for DNA polymerase-beta's substrate-induced conformational change

Structural information for mammalian DNA pol-beta combined with molecular and essential dynamics studies have provided atomistically detailed views of functionally important conformational rearrangements that occur during DNA repair and replication. This conformational closing before the chemical reaction is explored in this work as a function of the bound substrate. Anchors for our study are available in crystallographic structures of the DNA pol-beta in "open" (polymerase bound to gapped DNA) and "closed" (polymerase bound to gapped DNA and substrate, dCTP) forms; these different states have long been used to deduce that a large-scale conformational change may help the polymerase choose the correct nucleotide, and hence monitor DNA synthesis fidelity, through an "induced-fit" mechanism. However, the existence of open states with bound substrate and closed states without substrates suggest that substrate-induced conformational closing may be more subtle. Our dynamics simulations of two pol-beta/DNA systems (with/without substrates at the active site) reveal the large-scale closing motions of the thumb and 8-kDa subdomains in the presence of the correct substrate--leading to nearly perfect rearrangement of residues in the active site for the subsequent chemical step of nucleotidyl transfer--in contrast to an opening trend when the substrate is absent, leading to complete disassembly of the active site residues. These studies thus provide in silico evidence for the substrate-induced conformational rearrangements, as widely assumed based on a variety of crystallographic open and closed complexes. Further details gleaned from essential dynamics analyses clarify functionally relevant global motions of the polymerase-beta/DNA complex as required to prepare the system for the chemical reaction of nucleotide extension.     

Mechanism of DNA Methylation: The Double Role of DNA as a Substrate and as a Cofactor

Methylation of cytosine residues in the DNA is one of the most important epigenetic marks central to the control of differential expression of genes. We perform quantum mechanical calculations to investigate the catalytic mechanism of the bacterial HhaI DNA methyltransferase. We find that the enzyme nucleophile, Cys81, can attack C6 of cytosine only after it is deprotonated by the DNA phosphate group, a reaction facilitated by a bridging water molecule. This finding, which indicates that the DNA acts as both the substrate and the cofactor, can explain the total loss of activity observed in an analogous enzyme, thymidylate synthase, when the phosphate group of the substrate was removed. Furthermore, our results displaying the inability of the phosphate group to deprotonate the side chain of serine is in agreement with the total, or the large extent of, inactivity observed for the C81S mutant. In contrast to results from previous calculations, we find that the active site conserved residues, Glu119, Arg163, and Arg165, are crucial for catalysis. In addition, the enzyme-DNA adduct formation and the methyl transfer from the cofactor S-adenosyl-l-methionine are not concerted but proceed via stepwise mechanism. In many of the different steps of this methylation reaction, the transfer of a proton is found to be necessary. To render these processes possible, we find that several water molecules, found in the crystal structure, play an important role, acting as a bridge between the donating and accepting proton groups.     

Structural and Functional Analyses of β-Glucosidase 3B from Thermotoga neapolitana: A Thermostable Three-Domain Representative of Glycoside Hydrolase 3

Based on sequence and phylogenetic analyses, glycoside hydrolase (GH) family 3 can be divided into several clusters that differ in the length of their primary sequences. However, structural data on representatives of GH3 are still scarce, since only three of their structures are known and only one of them has been thoroughly characterized—that of an exohydrolase from barley. To allow a deeper structural understanding of the GH3 family, we have determined the crystal structure of the thermostable β-glucosidase from Thermotoga neapolitana, which has potentially important applications in environmentally friendly industrial biosynthesis at a resolution of 2.05 Å. Selected active-site mutants have been characterized kinetically, and the structure of the mutant D242A is presented at 2.1 Å resolution. Bgl3B from Th. neapolitana is the first example of a GH3 glucosidase with a three-domain structure. It is composed of an (α/β)₈ domain similar to a triose phosphate isomerase barrel, a five-stranded α/β sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain of unknown function. Remarkably, the direction of the second β-strand of the triose phosphate isomerase barrel domain is reversed, which has implications for the active-site shape. The active site, at the interface of domains 1 and 2, is much more open to solvent than the corresponding site in the structurally homologous enzyme from barley, and only the − 1 site is well defined. The structures, in combination with kinetic studies of active-site variants, allow the identification of essential catalytic residues (the nucleophile D242 and the acid/base E458), as well as other residues at the − 1 subsite, including D58 and W243, which, by mutagenesis, are shown to be important for substrate accommodation/interaction. The position of the fibronectin type III domain excludes a direct participation of this domain in the recognition of small substrates, although it may be involved in the anchoring of the enzyme on large polymeric substrates and in thermostability.