The Role of Tryptophan in π Interactions in Proteins: An Experimental Approach.

In proteins, the amino acids Phe, Tyr, and especially Trp are frequently involved in π interactions such as π-π, cation-π, and CH-π bonds. These interactions are often crucial for protein structure and protein-ligand binding. A powerful means to study these interactions is progressive fluorination of these aromatic residues to modulate the electrostatic component of the interaction. However, to date no protein expression platform is available to produce milligram amounts of proteins labeled with such fluorinated amino acids. Here, we present a Lactococcus lactis Trp auxotroph-based expression system for efficient incorporation (≥95%) of mono-, di-, tri-, and tetrafluorinated, as well as a methylated Trp analog. As a model protein we have chosen LmrR, a dimeric multidrug transcriptional repressor protein from L. lactis. LmrR binds aromatic drugs, like daunomycin and riboflavin, between Trp96 and Trp96' in the dimer interface. Progressive fluorination of Trp96 decreased the affinity for the drugs 6- to 70-fold, clearly establishing the importance of electrostatic π-π interactions for drug binding. Presteady state kinetic data of the LmrR-drug interaction support the enthalpic nature of the interaction, while high resolution crystal structures of the labeled protein-drug complexes provide for the first time a structural view of the progressive fluorination approach. The L. lactis expression system was also used to study the role of Trp68 in the binding of riboflavin by the membrane-bound riboflavin transport protein RibU from L. lactis. Progressive fluorination of Trp68 revealed a strong electrostatic component that contributed 15-20% to the total riboflavin-RibU binding energy.

Relationship of Agaricus bisporus mannose-binding protein to lectins with ß-trefoil fold.

Agaricus bisporus mannose-binding protein (Abmb) was discovered as part of the mushroom tyrosinase (PPO3) complex, but its function in the mushroom has remained obscure. The protein has a ß-trefoil structure that is common for Ricin-B-like lectins. Indeed, its closest structural homologs are the hemagglutinin components of botulinum toxin (HA-33) and the Ricin-B-like lectin from Clitocybe nebularis (CNL), both of which bind galactose, and actinohivin, a recently discovered mannose-binding lectin from actinomycetes. Here we show that Abmb is evolutionarily related to them, which are lectins with a ß-trefoil fold. We also show for the first time that Abmb can exhibit typical lectin agglutination activity but only when in the complex with mushroom tyrosinase. This is unexpected and unique because the two proteins are not evolutionarily related and have different activities. Lectin and tyrosinase major role in defense mechanism as well as Abmb and PPO3 gene regulation during the early stages of the development of mushroom fruiting bodies suggested that Abmb has likely a function in defense against bacterial infection and/or insect-induced damage.

Phenylthiourea Binding to Human Tyrosinase-Related Protein 1.

Tyrosinase-related protein 1 (TYRP1) is one of the three human melanogenic enzymes involved in the biosynthesis of melanin, a pigment responsible for the color of the skin, hair, and eyes. It shares high sequence identity with tyrosinase, but has two zinc ions in its active site rather than two copper ions as in tyrosinase. Typical tyrosinase inhibitors do not directly coordinate to the zinc ions of TYRP1. Here, we show, from an X-ray crystal structure determination, that phenylthiourea, a highly potent tyrosinase inhibitor, does neither coordinate the active site zinc ions, but binds differently from other structurally characterized TYRP1-inhibitor complexes. Its aromatic ring is directed outwards from the active site, apparently as a result from the absence of polar oxygen substituents that can take the position of water molecules bound in the active site. The compound binds via hydrophobic interactions, thereby blocking substrate access to the active site.

Functional MRI to Study Gait Impairment in Parkinson's Disease: a Systematic Review and Exploratory ALE Meta-Analysis.

PURPOSE OF REVIEW: Whilst gait impairment is a main cause for disability in Parkinson's disease (PD), its neural control remains poorly understood. We performed a systematic review and meta-analysis of neuroimaging studies of surrogate features of gait in PD. FINDINGS: Assessing the results from PET or SPECT scans after a period of actual walking as well as fMRI during mental imagery or virtual reality (VR) gait paradigms, we found a varying pattern of gait-related brain activity. Overall, a decrease in activation of the SMA during gait was found in PD compared to elderly controls. In addition, the meta-analysis showed that the most consistent gait-related activation was situated in the cerebellar locomotor region (CLR) in PD. Despite methodological heterogeneity, the combined neuroimaging studies of gait provide new insights into its neural control in PD, suggesting that CLR activation likely serves a compensatory role in locomotion.

ß-Xylosidases: Structural Diversity, Catalytic Mechanism, and Inhibition by Monosaccharides.

Xylan, a prominent component of cellulosic biomass, has a high potential for degradation into reducing sugars, and subsequent conversion into bioethanol. This process requires a range of xylanolytic enzymes. Among them, ß-xylosidases are crucial, because they hydrolyze more glycosidic bonds than any of the other xylanolytic enzymes. They also enhance the efficiency of the process by degrading xylooligosaccharides, which are potent inhibitors of other hemicellulose-/xylan-converting enzymes. On the other hand, the ß-xylosidase itself is also inhibited by monosaccharides that may be generated in high concentrations during the saccharification process. Structurally, ß-xylosidases are diverse enzymes with different substrate specificities and enzyme mechanisms. Here, we review the structural diversity and catalytic mechanisms of ß-xylosidases, and discuss their inhibition by monosaccharides.

Structure and Function of Human Tyrosinase and Tyrosinase-Related Proteins.

Melanin is the main pigment responsible for the color of human skin, hair and eye. Its biosynthesis requires three melanogenic enzymes, tyrosinase (TYR), and the tyrosinase-related proteins TYRP1 and TYRP2. The difficulty of isolating pure and homogeneous proteins from endogenous sources has hampered their study, and resulted in many contradictory findings regarding their physiological functions. In this review, we summarize recent advances on the structure and function of TYR and TYRPs by virtue of the crystal structure of human TYRP1, which is the first available structure of a mammalian melanogenic enzyme. This structure, combined with tyrosinase structures from other lower eukaryotes and mutagenesis studies of key active site residues, sheds light on the mechanism of TYR and TYRPs. Furthermore, a TYRP1-based homology model of TYR provides a high-quality platform to map and analyze albinism-related mutations, as well as the design of specific antimelanogenic compounds. Finally, we provide perspectives for future structure/function studies of TYR and TYRPs.

Reducing virulence of the human pathogen Burkholderia by altering the substrate specificity of the quorum-quenching acylase PvdQ.

The use of enzymes to interfere with quorum sensing represents an attractive strategy to fight bacterial infections. We used PvdQ, an effective quorum-quenching enzyme from Pseudomonas aeruginosa, as a template to generate an acylase able to effectively hydrolyze C8-HSL, the major communication molecule produced by the Burkholderia species. We discovered that the combination of two single mutations leading to variant PvdQ(Lα146W,Fß24Y) conferred high activity toward C8-HSL. Exogenous addition of PvdQ(Lα146W,Fß24Y) dramatically decreased the amount of C8-HSL present in Burkholderia cenocepacia cultures and inhibited a quorum sensing-associated phenotype. The efficacy of this PvdQ variant to combat infections in vivo was further confirmed by its ability to rescue Galleria mellonella larvae upon infection, demonstrating its potential as an effective agent toward Burkholderia infections. Kinetic analysis of the enzymatic activities toward 3-oxo-C12-L-HSL and C8-L-HSL corroborated a substrate switch. This work demonstrates the effectiveness of quorum-quenching acylases as potential novel antimicrobial drugs. In addition, we demonstrate that their substrate range can be easily switched, thereby paving the way to selectively target only specific bacterial species inside a complex microbial community.

Structure of Human Tyrosinase Related Protein†1 Reveals a Binuclear Zinc Active Site Important for Melanogenesis.

Tyrosinase-related protein 1 (TYRP1) is one of three tyrosinase-like glycoenzymes in human melanocytes that are key to the production of melanin, the compound responsible for the pigmentation of skin, eye, and hair. Difficulties with producing these enzymes in pure form have hampered the understanding of their activity and the effect of mutations that cause albinism and pigmentation disorders. Herein we show that the typical tyrosinase-like subdomain of TYRP1 contains two zinc ions in the active site instead of copper ions as found in tyrosinases, which explains why TYRP1 does not exhibit tyrosinase redox activity. In addition, the structures reveal for the first time that the Cys-rich subdomain, which is unique to vertebrate melanogenic proteins, has an epidermal growth factor-like fold and is tightly associated with the tyrosinase subdomain. Our structures suggest that most albinism-related mutations of TYRP1 affect its stability or activity.

Structural Basis of the Substrate Range and Enantioselectivity of Two (S)-Selective ω-Transaminases.

ω-Transaminases are enzymes that can introduce an amino group in industrially interesting compounds. We determined crystal structures of two (S)-selective ω-transaminases, one from Arthrobacter sp. (Ars-ωTA) and one from Bacillus megaterium (BM-ωTA), which have 95% identical sequences but somewhat different activity profiles. Substrate profiling measurements using a range of (R)- and (S)-substrates showed that both enzymes have a preference for substrates with large, flat cyclic side groups, for which the activity of BM-ωTA is generally somewhat higher. BM-ωTA has a preference for (S)-3,3-dimethyl-2-butylamine significantly stronger than that of Ars-ωTA, as well as a weaker enantiopreference for 1-cyclopropylethylamine. The crystal structures showed that, as expected for (S)-selective transaminases, both enzymes have the typical transaminase type I fold and have spacious active sites to accommodate largish substrates. A structure of BM-ωTA with bound (R)-α-methylbenzylamine explains the enzymes' preference for (S)-substrates. Site-directed mutagenesis experiments revealed that the presence of a tyrosine, instead of a cysteine, at position 60 increases the relative activities on several small substrates. A structure of Ars-ωTA with bound l-Ala revealed that the Arg442 side chain has been repositioned to bind the l-Ala carboxylate. Compared to the arginine switch residue in other transaminases, Arg442 is shifted by six residues in the amino acid sequence, which appears to be a consequence of extra loops near the active site that narrow the entrance to the active site.

A novel immune-tolerable and permeable lectin-like protein from mushroom Agaricus bisporus.

A lectin like protein designated as LSMT is recently discovered in Agaricus bisporus. The protein adopts very similar structure to Ricin-B like lectin from Clitocybe nebularis (CNL) and HA-33 from Clostridium botulinum (HA-33), which both recognize sugar molecules that decorate the surface of the epithelial cells of the intestine. A preliminary study in silico pointed out potential capability of LSMT to perform such biological activity. Following that hypothesis, we demonstrated that LSMT is indeed capable of penetrating out from a dialysis tube of the mice intestine origin. Furthermore, the protein appeared not to evoke the immune response upon introduction into mice, unlike its structural homologs. This is the first report on the biological implication of LSMT that might lead to its application.

Crystal structures of two Bacillus carboxylesterases with different enantioselectivities.

Naproxen esterase (NP) from Bacillus subtilis Thai I-8 is a carboxylesterase that catalyzes the enantioselective hydrolysis of naproxenmethylester to produce S-naproxen (E>200). It is a homolog of CesA (98% sequence identity) and CesB (64% identity), both produced by B. subtilis strain 168. CesB can be used for the enantioselective hydrolysis of 1,2-O-isopropylideneglycerol (solketal) esters (E>200 for IPG-caprylate). Crystal structures of NP and CesB, determined to a resolution of 1.75Å and 2.04Å, respectively, showed that both proteins have a canonical α/ß hydrolase fold with an extra N-terminal helix stabilizing the cap subdomain. The active site in both enzymes is located in a deep hydrophobic groove and includes the catalytic triad residues Ser130, His274, and Glu245. A product analog, presumably 2-(2-hydroxyethoxy)acetic acid, was bound in the NP active site. The enzymes have different enantioselectivities, which previously were shown to result from only a few amino acid substitutions in the cap domain. Modeling of a substrate in the active site of NP allowed explaining the different enantioselectivities. In addition, Ala156 may be a determinant of enantioselectivity as well, since its side chain appears to interfere with the binding of certain R-enantiomers in the active site of NP. However, the exchange route for substrate and product between the active site and the solvent is not obvious from the structures. Flexibility of the cap domain might facilitate such exchange. Interestingly, both carboxylesterases show higher structural similarity to meta-cleavage compound (MCP) hydrolases than to other α/ß hydrolase fold esterases.

X-ray crystallographic validation of structure predictions used in computational design for protein stabilization.

Protein engineering aimed at enhancing enzyme stability is increasingly supported by computational methods for calculation of mutant folding energies and for the design of disulfide bonds. To examine the accuracy of mutant structure predictions underlying these computational methods, crystal structures of thermostable limonene epoxide hydrolase variants obtained by computational library design were determined. Four different predicted effects indeed contributed to the obtained stabilization: (i) enhanced interactions between a flexible loop close to the N-terminus and the rest of the protein; (ii) improved interactions at the dimer interface; (iii) removal of unsatisfied hydrogen bonding groups; and (iv) introduction of additional positively charged groups at the surface. The structures of an eightfold and an elevenfold mutant showed that most mutations introduced the intended stabilizing interactions, and side-chain conformations were correctly predicted for 72-88% of the point mutations. However, mutations that introduced a disulfide bond in a flexible region had a larger influence on the backbone conformation than predicted. The enzyme active sites were unaltered, in agreement with the observed preservation of catalytic activities. The structures also revealed how a c-Myc tag, which was introduced for facile detection and purification, can reduce access to the active site and thereby lower the catalytic activity. Finally, sequence analysis showed that comprehensive mutant energy calculations discovered stabilizing mutations that are not proposed by the consensus or B-FIT methods.

Structural investigations into the stereochemistry and activity of a phenylalanine-2,3-aminomutase from Taxus chinensis.

Phenylalanine-2,3-aminomutase (PAM) from Taxus chinensis, a 4-methylidene-imidazole-5-one (MIO)-dependent enzyme, catalyzes the reversible conversion of (S)-α-phenylalanine into (R)-ß-phenylalanine via trans-cinnamic acid. The enzyme also catalyzes the direct addition of ammonia to trans-cinnamic acid, a reaction that can be used for the preparation of ß-amino acids, which occur as frequent constituents of bioactive compounds. Different hypotheses have been formulated to explain the stereochemistry of the PAM-catalyzed reaction, but structural evidence for these hypotheses is lacking. Furthermore, it remains unclear how the PAM MIO group is formed from the three-amino acid (A-S-G) sequence motif. For these reasons, we elucidated PAM three-dimensional (3D) structures with a bound (R)-ß-phenylalanine analogue and with bound trans-cinnamic acid. In addition, 3D structures of the (inactive) Y322A and N231A mutants of PAM were elucidated, which were found to be MIO-less. We conclude that the stereochemistry of the PAM-catalyzed reaction originates from the enzyme's ability to bind trans-cinnamic acid in two different orientations, with either the si,si face or the re,re face directed toward the MIO group, as evidenced by two distinct carboxylate binding modes. The results also suggest that the N231 side chain promotes MIO group formation by increasing the nucleophilicity of the G177 N atom through acidification of the amide proton.

Crystal structure and site-directed mutagenesis of 3-ketosteroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1 explain its catalytic mechanism.

3-Ketosteroid Δ(1)-dehydrogenases are FAD-dependent enzymes that catalyze the 1,2-desaturation of 3-ketosteroid substrates to initiate degradation of the steroid nucleus. Here we report the 2.0 Å resolution crystal structure of the 56-kDa enzyme from Rhodococcus erythropolis SQ1 (Δ(1)-KSTD1). The enzyme contains two domains: an FAD-binding domain and a catalytic domain, between which the active site is situated as evidenced by the 2.3 Å resolution structure of Δ(1)-KSTD1 in complex with the reaction product 1,4-androstadiene-3,17-dione. The active site contains four key residues: Tyr(119), Tyr(318), Tyr(487), and Gly(491). Modeling of the substrate 4-androstene-3,17-dione at the position of the product revealed its interactions with these residues and the FAD. The C1 and C2 atoms of the substrate are at reaction distance to the N5 atom of the isoalloxazine ring of FAD and the hydroxyl group of Tyr(318), respectively, whereas the C3 carbonyl group is at hydrogen bonding distance from the hydroxyl group of Tyr(487) and the backbone amide of Gly(491). Site-directed mutagenesis of the tyrosines to phenylalanines confirmed their importance for catalysis. The structural features and the kinetic properties of the mutants suggest a catalytic mechanism in which Tyr(487) and Gly(491) work in tandem to promote keto-enol tautomerization and increase the acidity of the C2 hydrogen atoms of the substrate. With assistance of Tyr(119), the general base Tyr(318) abstracts the axial ß-hydrogen from C2 as a proton, whereas the FAD accepts the axial α-hydrogen from the C1 atom of the substrate as a hydride ion.

Crystal structure of α-1,4-glucan lyase, a unique glycoside hydrolase family member with a novel catalytic mechanism.

α-1,4-Glucan lyase (EC 4.2.2.13) from the red seaweed Gracilariopsis lemaneiformis cleaves α-1,4-glucosidic linkages in glycogen, starch, and malto-oligosaccharides, yielding the keto-monosaccharide 1,5-anhydro-D-fructose. The enzyme belongs to glycoside hydrolase family 31 (GH31) but degrades starch via an elimination reaction instead of hydrolysis. The crystal structure shows that the enzyme, like GH31 hydrolases, contains a (ß/α)8-barrel catalytic domain with B and B' subdomains, an N-terminal domain N, and the C-terminal domains C and D. The N-terminal domain N of the lyase was found to bind a trisaccharide. Complexes of the enzyme with acarbose and 1-dexoynojirimycin and two different covalent glycosyl-enzyme intermediates obtained with fluorinated sugar analogues show that, like GH31 hydrolases, the aspartic acid residues Asp(553) and Asp(665) are the catalytic nucleophile and acid, respectively. However, as a unique feature, the catalytic nucleophile is in a position to act also as a base that abstracts a proton from the C2 carbon atom of the covalently bound subsite -1 glucosyl residue, thus explaining the unique lyase activity of the enzyme. One Glu to Val mutation in the active site of the homologous α-glucosidase from Sulfolobus solfataricus resulted in a shift from hydrolytic to lyase activity, demonstrating that a subtle amino acid difference can promote lyase activity in a GH31 hydrolase.

Structural and time-resolved mechanistic investigations of protein hydrolysis by the acidic proline-specific endoprotease from Aspergillus niger.

Proline-specific endoproteases have been successfully used in, for example, the in-situ degradation of gluten, the hydrolysis of bitter peptides, the reduction of haze during beer production, and the generation of peptides for mass spectroscopy and proteomics applications. Here we present the crystal structure of the extracellular proline-specific endoprotease from Aspergillus niger (AnPEP), a member of the S28 peptidase family with rarely observed true proline-specific endoprotease activity. Family S28 proteases have a conventional Ser-Asp-His catalytic triad, but their oxyanion-stabilizing hole shows a glutamic acid, an amino acid not previously observed in this role. Since these enzymes have an acidic pH optimum, the presence of a glutamic acid in the oxyanion hole may confine their activity to an acidic pH. Yet, considering the presence of the conventional catalytic triad, it is remarkable that the A. niger enzyme remains active down to pH 1.5. The determination of the primary cleavage site of cytochrome c along with molecular dynamics-assisted docking studies indicate that the active site pocket of AnPEP can accommodate a reverse turn of approximately 12 amino acids with proline at the S1 specificity pocket. Comparison with the structures of two S28-proline-specific exopeptidases reveals not only a more spacious active site cavity but also the absence of any putative binding sites for amino- and carboxyl-terminal residues as observed in the exopeptidases, explaining AnPEP's observed endoprotease activity.

Structural determinants of the ß-selectivity of a bacterial aminotransferase.

Chiral ß-amino acids occur as constituents of various natural and synthetic compounds with potentially useful bioactivities. The pyridoxal 5'-phosphate (PLP)-dependent S-selective transaminase from Mesorhizobium sp. strain LUK (MesAT) is a fold type I aminotransferase that can be used for the preparation of enantiopure ß-Phe and derivatives thereof. Using x-ray crystallography, we solved structures of MesAT in complex with (S)-ß-Phe, (R)-3-amino-5-methylhexanoic acid, 2-oxoglutarate, and the inhibitor 2-aminooxyacetic acid, which allowed us to unveil the molecular basis of the amino acid specificity and enantioselectivity of this enzyme. The binding pocket of the side chain of a ß-amino acid is located on the 3'-oxygen side of the PLP cofactor. The same binding pocket is utilized by MesAT to bind the α-carboxylate group of an α-amino acid. A ß-amino acid thus binds in a reverse orientation in the active site of MesAT compared with an α-amino acid. Such a binding mode has not been reported before for any PLP-dependent aminotransferase and shows that the active site of MesAT has specifically evolved to accommodate both ß- and α-amino acids.

Structure and catalytic mechanism of 3-ketosteroid-Delta4-(5α)-dehydrogenase from Rhodococcus jostii RHA1 genome.

3-Ketosteroid Δ4-(5α)-dehydrogenases (Δ4-(5α)-KSTDs) are enzymes that introduce a double bond between the C4 and C5 atoms of 3-keto-(5α)-steroids. Here we show that the ro05698 gene from Rhodococcus jostii RHA1 codes for a flavoprotein with Δ4-(5α)-KSTD activity. The 1.6 Å resolution crystal structure of the enzyme revealed three conserved residues (Tyr-319, Tyr-466, and Ser-468) in a pocket near the isoalloxazine ring system of the FAD co-factor. Site-directed mutagenesis of these residues confirmed that they are absolutely essential for catalytic activity. A crystal structure with bound product 4-androstene-3,17-dione showed that Ser-468 is in a position in which it can serve as the base abstracting the 4ß-proton from the C4 atom of the substrate. Ser-468 is assisted by Tyr-319, which possibly is involved in shuttling the proton to the solvent. Tyr-466 is at hydrogen bonding distance to the C3 oxygen atom of the substrate and can stabilize the keto-enol intermediate occurring during the reaction. Finally, the FAD N5 atom is in a position to be able to abstract the 5α-hydrogen of the substrate as a hydride ion. These features fully explain the reaction catalyzed by Δ4-(5α)-KSTDs.

Functional and structural characterization of α-(1->2) branching sucrase derived from DSR-E glucansucrase.

ΔN(123)-glucan-binding domain-catalytic domain 2 (ΔN(123)-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1→2) linkages. The crystal structure of the apo α-(1→2) branching sucrase ΔN(123)-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite -1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN(123)-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN(123)-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN(123)-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1→2) branches.