A H NMR study of the specificity of α-l-arabinofuranosidases on natural and unnatural substrates

Background

The detailed characterization of arabinoxylan-active enzymes, such as double-substituted xylan arabinofuranosidase activity, is still a challenging topic. Ad hoc chromogenic substrates are useful tools and can reveal subtle differences in enzymatic behavior. In this study, enzyme selectivity on natural substrates has been compared with enzyme selectivity towards aryl-glycosides. This has proven to be a suitable approach to understand how artificial substrates can be used to characterize arabinoxylan-active α-l-arabinofuranosidases (Abfs).

Methods

Real-time NMR using a range of artificial chromogenic, synthetic pseudo-natural and natural substrates was employed to determine the hydrolytic abilities and specificity of different Abfs.

Results

The way in which synthetic di-arabinofuranosylated substrates are hydrolyzed by Abfs mirrors the behavior of enzymes on natural arabinoxylo-oligosaccharide (AXOS). Family GH43 Abfs that are strictly specific for mono-substituted d-xylosyl moieties (AXH-m) do not hydrolyze synthetic di-arabinofuranosylated substrates, while those specific for di-substituted moieties (AXH-d) remove a single l-arabinofuranosyl (l-Araf) group. GH51 Abfs, which are supposedly AXH-m enzymes, can release l-Araf from disubstituted d-xylosyl moieties, when these are non-reducing terminal groups.

Conclusions and general significance

The present study reveals that although the activity of Abfs on artificial substrates can be quite different from that displayed on natural substrates, enzyme specificity is well conserved. This implies that carefully chosen artificial substrates bearing di-arabinofuranosyl d-xylosyl moieties are convenient tools to probe selectivity in new Abfs. Moreover, this study has further clarified the relative promiscuity of GH51 Abfs, which can apparently hydrolyze terminal disubstitutions in AXOS, albeit less efficiently than mono-substituted motifs.     

Enzyme inhibitor modeling with TpZn complexes of functional hydroxamates and oximates

Salicylhydroxamic acid reacts with the enzyme model Tp^Ph,MeZn-OH to form the O,O-chelating hydroxamate complex 1. The hydrogen bonding capacity of zinc enzyme bound hydroxamates is reproduced by cocrystallization of two molecules if 1 with two molecules of methanol and by cocrystallization of one molecule of Tp^Ph,MeZn-acetohydroxamate with one molecule of 3-phenyl-5-methylpyrazole. The complex formed from Tp^Ph,MeZn-OH and N-tosylproline hydroxamic acid, according to its spectra, contains the hydroxamate as an N,N-chelating ligand. In contrast, the oximate derived from pyruvic aldehyde does not act as a chelating ligand, but is monodentate via the oximate oxygen.     

Crystal Structure and Characterization of the Glycoside Hydrolase Family 62 α-l-Arabinofuranosidase from Streptomyces coelicolor

α-l-Arabinofuranosidase, which belongs to the glycoside hydrolase family 62 (GH62), hydrolyzes arabinoxylan but not arabinan or arabinogalactan. The crystal structures of several α-l-arabinofuranosidases have been determined, although the structures, catalytic mechanisms, and substrate specificities of GH62 enzymes remain unclear. To evaluate the substrate specificity of a GH62 enzyme, we determined the crystal structure of α-l-arabinofuranosidase, which comprises a carbohydrate-binding module family 13 domain at its N terminus and a catalytic domain at its C terminus, from Streptomyces coelicolor. The catalytic domain was a five-bladed β-propeller consisting of five radially oriented anti-parallel β-sheets. Sugar complex structures with l-arabinose, xylotriose, and xylohexaose revealed five subsites in the catalytic cleft and an l-arabinose-binding pocket at the bottom of the cleft. The entire structure of this GH62 family enzyme was very similar to that of glycoside hydrolase 43 family enzymes, and the catalytically important acidic residues found in family 43 enzymes were conserved in GH62. Mutagenesis studies revealed that Asp²⁰² and Glu³⁶¹ were catalytic residues, and Trp²⁷⁰, Tyr⁴⁶¹, and Asn⁴⁶² were involved in the substrate-binding site for discriminating the substrate structures. In particular, hydrogen bonding between Asn⁴⁶² and xylose at the nonreducing end subsite +2 was important for the higher activity of substituted arabinofuranosyl residues than that for terminal arabinofuranoses.     

Cysteine as a Modulator Residue in the Active Site of Xenobiotic Reductase A: A Structural, Thermodynamic and Kinetic Study

Xenobiotic reductase A (XenA) from Pseudomonas putida 86 catalyzes the NADH/NADPH-dependent reduction of various substrates, including 2-cyclohexenone and 8-hydroxycoumarin. XenA is a member of the old yellow enzyme (OYE) family of flavoproteins and is structurally and functionally similar to other bacterial members of this enzyme class. A characteristic feature of XenA is the presence of a cysteine residue (Cys25) in the active site, where in most members of the OYE family a threonine residue is found that modulates the reduction potential of the FMN/FMNH^- couple. We investigated the role of Cys25 by studying two variants in which the residue has been exchanged for a serine and an alanine residue. While the exchange against alanine has a remarkably small effect on the reduction potential, the reactivity and the structure of XenA, the exchange against serine increases the reduction potential by +82 mV, increases the rate constant of the reductive half-reaction and decreases the rate constant in the oxidative half-reaction. We determined six crystal structures at high to true atomic resolution (d_min 1.03-1.80 Å) of the three XenA variants with and without the substrate coumarin bound in the active site. The atomic resolution structure of XenA in complex with coumarin reveals a compressed active site geometry in which the isoalloxazine ring is sandwiched between coumarin and the protein backbone. The structures further reveal that the conformation of the active site and substrate interactions are preserved in the two variants, indicating that the observed changes are due to local effects only. We propose that Cys25 and the residues in its place determine which of the two half-reactions is rate limiting, depending on the substrate couple. This might help to explain why the genome of Pseudomonas putida encodes multiple xenobiotic reductases containing either cysteine, threonine or alanine in the active site.     

Old Yellow Enzymes Protect against Acrolein Toxicity in the Yeast Saccharomyces cerevisiae

Acrolein is a ubiquitous reactive aldehyde which is formed as a product of lipid peroxidation in biological systems. In this present study, we screened the complete set of viable deletion strains in Saccharomyces cerevisiae for sensitivity to acrolein to identify cell functions involved in resistance to reactive aldehydes. We identified 128 mutants whose gene products are localized throughout the cell. Acrolein-sensitive mutants were distributed among most major biological processes but particularly affected gene expression, metabolism, and cellular signaling. Surprisingly, the screen did not identify any antioxidants or similar stress-protective molecules, indicating that acrolein toxicity may not be mediated via reactive oxygen species. Most strikingly, a mutant lacking an old yellow enzyme (OYE2) was identified as being acrolein sensitive. Old yellow enzymes are known to reduce α,β-unsaturated carbonyl compounds in vitro, but their physiological roles have remained uncertain. We show that mutants lacking OYE2, but not OYE3, are sensitive to acrolein, and overexpression of both isoenzymes increases acrolein tolerance. Our data indicate that OYE2 is required for basal levels of tolerance, whereas OYE3 expression is particularly induced following acrolein stress. Despite the range of α,β-unsaturated carbonyl compounds that have been identified as substrates of old yellow enzymes in vitro, we show that old yellow enzymes specifically mediate resistance to small α,β-unsaturated carbonyl compounds, such as acrolein, in vivo.     

Crystal Structures of Ethanolamine Ammonia-lyase Complexed with Coenzyme B12 Analogs and Substrates

N-terminal truncation of the Escherichia coli ethanolamine ammonia-lyase β-subunit does not affect the catalytic properties of the enzyme (Akita, K., Hieda, N., Baba, N., Kawaguchi, S., Sakamoto, H., Nakanishi, Y., Yamanishi, M., Mori, K., and Toraya, T. (2010) J. Biochem. 147, 83–93). The binary complex of the truncated enzyme with cyanocobalamin and the ternary complex with cyanocobalamin or adeninylpentylcobalamin and substrates were crystallized, and their x-ray structures were analyzed. The enzyme exists as a trimer of the (αβ)₂ dimer. The active site is in the (β/α)₈ barrel of the α-subunit; the β-subunit covers the lower part of the cobalamin that is bound in the interface of the α- and β-subunits. The structure complexed with adeninylpentylcobalamin revealed the presence of an adenine ring-binding pocket in the enzyme that accommodates the adenine moiety through a hydrogen bond network. The substrate is bound by six hydrogen bonds with active-site residues. Argα¹⁶⁰ contributes to substrate binding most likely by hydrogen bonding with the O1 atom. The modeling study implies that marked angular strains and tensile forces induced by tight enzyme-coenzyme interactions are responsible for breaking the coenzyme Co–C bond. The coenzyme adenosyl radical in the productive conformation was modeled by superimposing its adenine ring on the adenine ring-binding site followed by ribosyl rotation around the N-glycosidic bond. A major structural change upon substrate binding was not observed with this particular enzyme. Gluα²⁸⁷, one of the substrate-binding residues, has a direct contact with the ribose group of the modeled adenosylcobalamin, which may contribute to the substrate-induced additional labilization of the Co–C bond.     

Crystal structure of bacterial morphinone reductase and properties of the C191A mutant enzyme

The crystal structure of the NADH-dependent bacterial flavoenzyme morphinone reductase (MR) has been determined at 2.2-A resolution in complex with the oxidizing substrate codeinone. The structure reveals a dimeric enzyme comprising two 8-fold beta/alpha barrel domains, each bound to FMN, and a subunit folding topology and mode of flavin-binding similar to that found in Old Yellow Enzyme (OYE) and pentaerythritol tetranitrate (PETN) reductase. The subunit interface of MR is formed by interactions from an N-terminal beta strand and helices 2 and 8 of the barrel domain and is different to that seen in OYE. The active site structures of MR, OYE, and PETN reductase are highly conserved reflecting the ability of these enzymes to catalyze "generic" reactions such as the reduction of 2-cyclohexenone. A region of polypeptide presumed to define the reducing coenzyme specificity is identified by comparison of the MR structure (NADH-dependent) with that of PETN reductase (NADPH-dependent). The active site acid identified in OYE (Tyr-196) and conserved in PETN reductase (Tyr-186) is replaced by Cys-191 in MR. Mutagenesis studies have established that Cys-191 does not act as a crucial acid in the mechanism of reduction of the olefinic bond found in 2-cyclohexenone and codeinone.     

Divergence of cofactor recognition across evolution: coenzyme A binding in a prokaryotic arylamine N-acetyltransferase

Arylamine N-acetyltransferase (NAT) enzymes are widespread in nature. They serve to acetylate xenobiotics and/or endogenous substrates using acetyl coenzyme A (CoA) as a cofactor. Conservation of the architecture of the NAT enzyme family from mammals to bacteria has been demonstrated by a series of prokaryotic NAT structures, together with the recently reported structure of human NAT1. We report here the cloning, purification, kinetic characterisation and crystallographic structure determination of NAT from Mycobacterium marinum, a close relative of the pathogenic Mycobacterium tuberculosis. We have also determined the structure of M. marinum NAT in complex with CoA, shedding the first light on cofactor recognition in prokaryotic NATs. Surprisingly, the principal CoA recognition site in M. marinum NAT is located some 30 Å from the site of CoA recognition in the recently deposited structure of human NAT2 bound to CoA. The structure explains the Ping-Pong Bi-Bi reaction mechanism of NAT enzymes and suggests mechanisms by which the acetylated enzyme intermediate may be protected. Recognition of CoA in a much wider groove in prokaryotic NATs suggests that this subfamily may accommodate larger substrates than is the case for human NATs and may assist in the identification of potential endogenous substrates. It also suggests the cofactor-binding site as a unique subsite to target in drug design directed against NAT in mycobacteria.     

Phylogenetic analyses provide the first insights into the evolution of OVATE family proteins in land plants

Background and Aims

The OVATE gene encodes a nuclear-localized regulatory protein belonging to a distinct family of plant-specific proteins known as the OVATE family proteins (OFPs). OVATE was first identified as a key regulator of fruit shape in tomato, with nonsense mutants displaying pear-shaped fruits. However, the role of OFPs in plant development has been poorly characterized.

Methods

Public databases were searched and a total of 265 putative OVATE protein sequences were identified from 13 sequenced plant genomes that represent the major evolutionary lineages of land plants. A phylogenetic analysis was conducted based on the alignment of the conserved OVATE domain from these 13 selected plant genomes. The expression patterns of tomato SlOFP genes were analysed via quantitative real-time PCR. The pattern of OVATE gene duplication resulting in the expansion of the gene family was determined in arabidopsis, rice and tomato.

Key Results

Genes for OFPs were found to be present in all the sampled land plant genomes, including the early-diverged lineages, mosses and lycophytes. Phylogenetic analysis based on the amino acid sequences of the conserved OVATE domain defined 11 sub-groups of OFPs in angiosperms. Different evolutionary mechanisms are proposed for OVATE family evolution, namely conserved evolution and divergent expansion. Characterization of the AtOFP family in arabidopsis, the OsOFP family in rice and the SlOFP family in tomato provided further details regarding the evolutionary framework and revealed a major contribution of tandem and segmental duplications towards expansion of the OVATE gene family.

Conclusions

This first genome-wide survey on OFPs provides new insights into the evolution of the OVATE protein family and establishes a solid base for future functional genomics studies on this important but poorly characterized regulatory protein family in plants.     

Cyclin-dependent kinase 5 phosphorylates mammalian HMGB1 protein only if acetylated

Old yellow enzyme (OYE) is an NADPH oxidoreductase capable of reducing a variety of compounds. It contains flavin mononucleotide (FMN) as a prosthetic group. A ternary complex structure of OYE from Trypanosoma cruzi (TcOYE) with FMN and one of the substrates, p-hydroxybenzaldehyde, shows a striking movement around the active site upon binding of the substrate. From a structural comparison of other OYE complexed with 12-oxophytodienoate, we have constructed a complex structure with another substrate, prostaglandin H(2) (PGH(2)), to provide a proposed stereoselective reaction mechanism for the reduction of PGH(2) to prostaglandin F(2α) by TcOYE.     

Oxidation of alkyl nitronates catalyzed by 2-nitropropane dioxygenase from Hansenula mrakii

2-Nitropropane dioxygenase from Hansenula mrakii was expressed in Escherichia coli cells and purified in active and stable form using 60% saturation of ammonium sulfate and a single chromatographic step onto a DEAE column. MALDI-TOF mass spectrometric and spectrophotometric analyses of the flavin extracted by heat or acid denaturation of the enzyme indicated that FMN, and not FAD as erroneously reported previously, is present in a 1:1 stoichiometry with the protein. Inductively coupled plasma mass spectrometric analysis of the enzyme established that H. mrakii 2-nitropropane dioxygenase contains negligible amounts of iron, manganese, zinc, and copper ions, which are not catalytically relevant. Anaerobic substrate reduction and kinetic data using a Clark oxygen electrode to measure rates of oxygen consumption indicated that the enzyme is active on a broad range of alkyl nitronates, with a marked preference for unbranched substrates over propyl-2-nitronate. Interestingly, the enzyme reacts poorly, if at all, with nitroalkanes, as suggested by lack of both anaerobic reduction of the enzyme-bound flavin and consumption of oxygen with nitroethane, nitrobutane, and 2-nitropropane. Finally, both the tight binding of sulfite (K_d = 90 μM, at pH 8 and 15 °C) to the enzyme and the formation of the anionic flavosemiquinone upon anaerobic incubation with alkyl nitronates are consistent with the presence of a positively charged group in proximity of the N(1)C(2)O atoms of the FMN cofactor.     

Characterization of the Staphylococcus aureus rRNA Methyltransferase Encoded by orfX,the Gene Containing the Staphylococcal Chromosome Cassette mec (SCCmec) Insertion Site

The gene orfX is conserved among all staphylococci, and its complete sequence is maintained upon insertion of the staphylococcal chromosome cassette mec (SCCmec) genomic island, containing the gene encoding resistance to β-lactam antibiotics (mecA), into its C terminus. The function of OrfX has not been determined. We show that OrfX was constitutively produced during growth, that orfX could be inactivated without altering bacterial growth, and that insertion of SCCmec did not alter gene expression. We solved the crystal structure of OrfX at 1.7 Å and found that it belongs to the S-adenosyl-l-methionine (AdoMet)-dependent α/β-knot superfamily of SPOUT methyltransferases (MTases), with a high structural homology to YbeA, the gene product of the Escherichia coli 70 S ribosomal MTase RlmH. MTase activity was confirmed by demonstrating the OrfX-dependent methylation of the Staphylococcus aureus 70 S ribosome. When OrfX was crystallized in the presence of its AdoMet substrate, we found that each monomer of the homodimeric structure bound AdoMet in its active site. Solution studies using isothermal titration calorimetry confirmed that each monomer bound AdoMet but with different binding affinities (K_d = 52 ± 0.4 and 606 ± 2 μm). In addition, the structure shows that the AdoMet-binding pocket, formed by a deep trefoil knot, contains a bound phosphate molecule, which is the likely nucleotide methylation site. This study represents the first characterization of a staphylococcal ribosomal MTase and provides the first crystal structure of a member of the α/β-knot superfamily of SPOUT MTases in the RlmH or COG1576 family with bound AdoMet.     

The 1.3 A crystal structure of the flavoprotein YqjM reveals a novel class of Old Yellow Enzymes

Here we report the crystal structure of YqjM, a homolog of Old Yellow Enzyme (OYE) that is involved in the oxidative stress response of Bacillus subtilis. In addition to the oxidized and reduced enzyme form, the structures of complexes with p-hydroxybenzaldehyde and p-nitrophenol, respectively, were solved. As for other OYE family members, YqjM folds into a (alpha/beta)8-barrel and has one molecule of flavin mononucleotide bound non-covalently at the COOH termini of the beta-sheet. Most of the interactions that control the electronic properties of the flavin mononucleotide cofactor are conserved within the OYE family. However, in contrast to all members of the OYE family characterized to date, YqjM exhibits several unique structural features. For example, the enzyme exists as a homotetramer that is assembled as a dimer of catalytically dependent dimers. Moreover, the protein displays a shared active site architecture where an arginine finger (Arg336) at the COOH terminus of one monomer extends into the active site of the adjacent monomer and is directly involved in substrate recognition. Another remarkable difference in the binding of the ligand in YqjM is represented by the contribution of the NH2-terminal Tyr28 instead of a COOH-terminal tyrosine in OYE and its homologs. The structural information led to a specific data base search from which a new class of OYE oxidoreductases was identified that exhibits a strict conservation of active site residues, which are critical for this subfamily, most notably Cys26, Tyr28, Lys109, and Arg336. Therefore, YqjM is the first representative of a new bacterial subfamily of OYE homologs.     

The Sequence Dependence of Human Nucleotide Excision Repair Efficiencies of Benzo[a]pyrene-derived DNA Lesions: Insights into the Structural Factors that Favor Dual Incisions

Nucleotide excision repair (NER) is a vital cellular defense system against carcinogen-DNA adducts, which, if not repaired, can initiate cancer development. The structural features of bulky DNA lesions that account for differences in NER efficiencies in mammalian cells are not well understood. In vivo, the predominant DNA adduct derived from metabolically activated benzo[a]pyrene (BP), a prominent environmental carcinogen, is the 10S (+)-trans-anti-[BP]-N²-dG adduct (G*), which resides in the B-DNA minor groove 5′-oriented along the modified strand. We have compared the structural distortions in double-stranded DNA, imposed by this adduct, in the different sequence contexts 5′-…CGG*C…, 5′-…CG*GC…, 5′-…CIG*C… (I is 2′-deoxyinosine), and 5′-…CG*C…. On the basis of electrophoretic mobilities, all duplexes manifest moderate bends, except the 5′-…CGG*C…duplex, which exhibits an anomalous, slow mobility attributed to a pronounced flexible kink at the site of the lesion. This kink, resulting from steric hindrance between the 5′-flanking guanine amino group and the BP aromatic rings, both positioned in the minor groove, is abolished in the 5′-…CIG*C…duplex (the 2′-deoxyinosine group, I, lacks this amino group). In contrast, the sequence-isomeric 5′-…CG*GC…duplex exhibits only a moderate bend, but displays a remarkably increased opening rate at the 5′-flanking base pair of G*, indicating a significant destabilization of Watson-Crick hydrogen bonding. The NER dual incision product yields were compared for these different sequences embedded in otherwise identical 135-mer duplexes in cell-free human HeLa extracts. The yields of excision products varied by a factor of as much as ∼ 4 in the order 5′-...CG*GC…> 5′...CGG*C…≥ 5′...CIG*C…≥ 5′-…CG*C…. Overall, destabilized Watson-Crick hydrogen bonding, manifested in the 5′-...CG*GC...duplex, elicits the most significant NER response, while the flexible kink displayed in the sequence-isomeric 5′-...CGG*C...duplex represents a less significant signal in this series of substrates. These results demonstrate that the identical lesion can be repaired with markedly variable efficiency in different local sequence contexts that differentially alter the structural features of the DNA duplex around the lesion site.     

Co-Factor Binding Confers Substrate Specificity to Xylose Reductase from Debaryomyces hansenii

Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards “non-substrate” sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (K_d∼5.0–10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4–5 fold molar excess. Comparison of K_d values with K_m values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.     

Characterization of YqjM,an Old Yellow Enzyme homolog from Bacillus subtilis involved in the oxidative stress response

In this paper, we demonstrate that a protein from Bacillus subtilis (YqjM) shares many characteristic biochemical properties with the homologous yeast Old Yellow Enzyme (OYE); the enzyme binds FMN tightly but noncovalently, preferentially uses NADPH as a source of reducing equivalents, and forms charge transfer complexes with phenolic compounds such as p-hydroxybenzaldehyde. Like yeast OYE and other members of the family, YqjM catalyzes the reduction of the double bond of an array of alpha,beta-unsaturated aldehydes and ketones including nitroester and nitroaromatic compounds. Although yeast OYE was the first member of this family to be discovered in 1933 and was the first flavoenzyme ever to be isolated, the physiological role of the family still remains obscure. The finding that alpha,beta-unsaturated compounds are substrates provoked speculation that the OYE family might be involved in reductive degradation of xenobiotics or lipid peroxidation products. Here, for the first time, we demonstrate on the protein level that whereas YqjM shows a basal level of expression in B. subtilis, the addition of the toxic xenobiotic, trinitrotoluene, leads to a rapid induction of the protein in vivo denoting a role in detoxification. Moreover, we show that YqjM is rapidly induced in response to oxidative stress as exerted by hydrogen peroxide, demonstrating a potential physiological role for this enigmatic class of proteins.     

First structures of an active bacterial tyrosinase reveal copper plasticity

Tyrosinase is a member of the type 3 copper enzyme family that is involved in the production of melanin in a wide range of organisms. The crystal structures of a tyrosinase from Bacillus megaterium were determined at a resolution of 2.0-2.3 Å. The enzyme crystallized as a dimer in the asymmetric unit and was shown to be active in crystal. The overall monomeric structure is similar to that of the monomer of the previously determined tyrosinase from Streptomyces castaneoglobisporus, but it does not contain an accessory Cu-binding “caddie” protein. Two Cu(II) ions, serving as the major cofactors within the active site, are coordinated by six conserved histidine residues. However, determination of structures under different conditions shows varying occupancies and positions of the copper ions. This apparent mobility in copper binding modes indicates that there is a pathway by which copper is accumulated or lost by the enzyme. Additionally, we suggest that residues R209 and V218, situated in a second shell of residues surrounding the active site, play a role in substrate binding orientation based on their flexibility and position. The determination of a structure with the inhibitor kojic acid, the first tyrosinase structure with a bound ligand, revealed additional residues involved in the positioning of substrates in the active site. Comparison of wild-type structures with the structure of the site-specific variant R209H, which possesses a higher monophenolase/diphenolase activity ratio, lends further support to a previously suggested mechanism by which monophenolic substrates dock mainly to CuA.     

Exploring the active site of tripeptidyl-peptidase II through studies of pH dependence of reaction kinetics

Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine protease which forms a large enzyme complex (> 4 MDa). It is considered a potential drug target due to its involvement in specific physiological processes. However, information is scarce concerning the kinetic characteristics of TPP II and its active site features, which are important for design of efficient inhibitors. To amend this, we probed the active site by determining the pH dependence of TPP II catalysis. Access to pure enzyme is a prerequisite for kinetic investigations and herein we introduce the first efficient purification system for heterologously expressed mammalian TPP II. The pH dependence of kinetic parameters for hydrolysis of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was determined for murine, human and Drosophila melanogaster TPP II as well as mutant variants thereof. The investigation demonstrated that TPP II, in contrast to subtilisin, has a bell-shaped pH dependence of k_cat^app/K_M probably due to deprotonation of the N-terminal amino group of the substrate at higher pH. Since both the K_M and k_cat^app are lower for cleavage of AAA-pNA than for AAF-pNA we propose that the former can bind non-productively to the active site of the enzyme, a phenomenon previously observed with some substrates for subtilisin. Two mutant variants, H267A and D387G, showed bell-shaped pH-dependence of k_cat^app, possibly due to an impaired protonation of the leaving group. This work reveals previously unknown differences between TPP II orthologues and subtilisin as well as features that might be conserved within the entire family of subtilisin-like serine peptidases.     

Crystal structures of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello-oligomers show high flexibility in the substrate binding

Cellobiohydrolase from Melanocarpus albomyces (Cel7B) is a thermostable, single-module, cellulose-degrading enzyme. It has relatively low catalytic activity under normal temperatures, which allows structural studies of the binding of unmodified substrates to the native enzyme. In this study, we have determined the crystal structure of native Ma Cel7B free and in complex with three different cello-oligomers: cellobiose (Glc₂), cellotriose (Glc₃), and cellotetraose (Glc₄), at high resolution (1.6–2.1 Å). In each case, four molecules were found in the asymmetric unit, which provided 12 different complex structures. The overall fold of the enzyme is characteristic of a glycoside hydrolase family 7 cellobiohydrolase, where the loops extending from the core β-sandwich structure form a long tunnel composed of multiple subsites for the binding of the glycosyl units of a cellulose chain. The catalytic residues at the reducing end of the tunnel are conserved, and the mechanism is expected to be retaining similarly to the other family 7 members. The oligosaccharides in different complex structures occupied different subsite sets, which partly overlapped and ranged from −5 to +2. In four cellotriose and one cellotetraose complex structures, the cello-oligosaccharide also spanned over the cleavage site (−1/+1). There were surprisingly large variations in the amino acid side chain conformations and in the positions of glycosyl units in the different cello-oligomer complexes, particularly at subsites near the catalytic site. However, in each complex structure, all glycosyl residues were in the chair (⁴C₁) conformation. Implications in relation to the complex structures with respect to the reaction mechanism are discussed.