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Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a Parkinson disease-associated, putative cysteine protease found abundantly and selectively expressed in neurons. The crystal structure of apo UCHL1 showed that the active-site residues are not aligned in a canonical form, with the nucleophilic cysteine being 7.7 [Angstrom] from the general base histidine, an arrangement consistent with an inactive form of the enzyme. Here we report the crystal structures of the wild type and two Parkinson disease-associated variants of the enzyme, S18Y and 193M, bound to a ubiquitin-based suicide substrate, ubiquitin vinyl methyl ester. These structures reveal that ubiquitin vinyl methyl ester binds primarily at two sites on the enzyme, with its carboxy terminus at the active site and with its amino-terminal D-hairpin at the distal site--a surface-exposed hydrophobic crevice 17 [Angstrom] away from the active site. Binding at the distal site initiates a cascade of side-chain movements in the enzyme that starts at a highly conserved, surface-exposed phenylalanine and is relayed to the active site resulting in the reorientation and proximal placement of the general base within 4 A of the catalytic cysteine, an arrangement found in productive cysteine proteases. Mutation of the distal-site, surface-exposed phenylalanine to alanine reduces ubiquitin binding and severely impairs the catalytic activity of the enzyme. These results suggest that the activity of UCHL1 may be regulated by its own substrate. deubiquitinating enzyme | enzyme suicide substrate complex | neurodegeneration | ubiquitination doi: 10.1073/pnas.0910870107

We report a catalytically promiscuous enzyme able to efficiently promote the hydrolysis of six different substrate classes. Originally assigned as a phosphonate monoester hydrolase (PMH) this enzyme exhibits substantial second-order rate accelerations (([k.sub.cat]/[K.sub.M])/ [k.sub.w]), ranging from [10.sup.7] to as high as [10.sup.19], for the hydrolyses of phosphate mono-, di-, and triesters, phosphonate monoesters, sulfate monoesters, and sulfonate monoesters. This substrate collection encompasses a range of substrate charges between 0 and -2, transition states of a different nature, and involves attack at two different reaction centers (P and S). Intrinsic reactivities (half-lives) range from 200 days to [10.sup.5] years under near neutrality. The substantial rate accelerations for a set of relatively difficult reactions suggest that efficient catalysis is not necessarily limited to efficient stabilization of just one transition state. The crystal structure of PMH identifies it as a member of the alkaline phosphatase superfamily. PMH encompasses four of the native activities previously observed in this superfamily and extends its repertoire by two further activities, one of which, sulfonate monoesterase, has not been observed previously for a natural enzyme. PMH is thus one of the most promiscuous hydrolases described to date. The functional links between superfamily activities can be presumed to have played a role in functional evolution by gene duplication. catalytic promiscuity | evolution | mechanism | sulfatase | superfamily www.pnas.org/cgi/doi/10.1073/pnas.0903951107

Nucleoside 5'-diphosphate-X hydrolases are interesting enzymes to study due to their varied activities and structure-function relationships and the roles they play in the disposal, assimilation, and modulation of the effects of their substrates. Few of these enzymes with a preference for CDP-alcohols are known. In Yersinia intermedia suspensions prepared from cultures on Columbia agar with 5% sheep blood, we found a CDP-alcohol hydrolase liberated to Triton X-100-containing medium. Growth at 25[degrees]C was deemed optimum in terms of the enzyme-activity yield. The purified enzyme also displayed 5'-nucleotidase, UDP-sugar hydrolase, and dinucleoside-polyphosphate hydrolase activities. It was identified as the protein product ([UshA.sub.Yi]) of the Y. intermedia ushA gene ([ushA.sub.Yi]) by its peptide mass fingerprint and by PCR cloning and expression to yield active enzyme. All those activities, except CDP-alcohol hydrolase, have been shown to be the properties of UshA of Escherichia coli ([UshA.sub.Ec]). Therefore, [UshA.sub.Ec] was expressed from an appropriate plasmid and tested for CDP-alcohol hydrolase activity. [UshA.sub.Ec] and [UshA.sub.Yi] behaved similarly. Besides being the first study of a UshA enzyme in the genus Yersinia, this work adds CDP-alcohol hydrolase to the spectrum of UshA activities and offers a novel perspective on these proteins, which are viewed here for the first time as highly efficient enzymes with [k.sub.cat]/[K.sub.m] ratios near the theoretical maximum level of catalytic activities. The results are discussed in the light of the known structures of [UshA.sub.Ec] conformers and the respective homology models constructed for [UshA.sub.Yi], and also in relation to possible biological functions. Interestingly, every Yersinia species with a sequenced genome contains an intact ushA gene, except Y. pestis, which in all its sequenced biovars contains a ushA gene inactivated by frameshift mutations.

A study was conducted to show that the equilibrium and kinetic properties of association and dissociation of the cofactor by Hs-SAHH and Tc-SAHH are qualitatively similar. Both enzymes exhibit the dimer of dimers structure in which the homotetramer is made up of two pairs of monomers and within each pair of monomers, a C-terminal segment from each partner penetrates into the other partner subunit and forms part of the cofactor binding site.

An orthologue of a retro-Claisenase from Rhodococcus sp. in the cyanobacterium Anabaena PCC 7120 was identified by genome mining. Studies of the enzyme revealed an additional rare example of an orthologue among the crotonase-related enzymes, which is not dependent on the ligation of its substrate to coenzyme A for substrate recognition.

The mechanism of ubiquitin C-terminal hydrolase L1 (UCH-L1), a UCH that is implicated in Parkinson's disease, is analyzed. The isotope effects for association of UCH-L1 with transition-state analogue aldehyde indicate that an alternative mechanistic pathway is available to UCH-L1 involving general base-catalyzed attack of Cys-SH by His-Im.

Kinetic, crystallographic and mutagenesis studies were conducted to identify critical residues to gain insight into the reaction mechanism of the enzyme and its apparent lack of cyclohydrolase activity. Single mutants Thr57Lys and Tyr98Gln and double mutant T57K/Y98Q were prepared to test the hypothesis that the lack of cyclohydrolase activity in y 5,10-methylenetertrahdrofolate dehydrogenase (MTD) was due to the substitution of a conserved Lys/Gln pair found in bifunctional MTDs.

A study investigates the kinetic resolution of a range of methyl-substituted 1-oxaspirol[2.5]octanes by yeast epoxide hydrolase (YEH) from Rhodotorula glutinis. The structural determinants of substrate specificity and stereoselectivity of YEH toward these substrates appears to be the configuration of the epoxide ring and the substitution pattern of the cyclohexane ring.

Directed evolution using iterative saturation mutagenesis (ISM) is tested as a means to engineer limonene epoxide hydrolase (LEH) mutants from Rhodococcus erythropolis DCL 14, known to be an exceptional epoxide hydrolase (EH). The findings contributed to the generalization of ISM as a fast and reliable method for protein engineering and proposed a simple model based on molecular dynamics simulations to explain some of the stereoselective consequences of the observed mutations.

The first functional and structural characterization of the inosine-guanosine-specific nucleoside hydrolase (NH) from Trypanosoma brucei brucei is reported. The study identified nanomolar iminoribitol-based inhibitors which could be important lead compounds for the development of novel therapeutic strategies against trypanosomal diseases.

X-ray structures of S-adenosylhomocysteine hydrolases (SAHHs) are used for applying free energy simulations using the MM-GBSA approach for predicting residues important for the differential cofactor binding properties of human and trypanosomal SAHHs (Hs-SAHH and Tc-SAHH), within the cofactor [NAD.sup.+]/NADH binding site. The identical residues in two orthologous proteins have given different binding free energy contributions, which have suggested that comparative studies of homologous proteins should examine not only different residues but also identical residues in these proteins.

Kinetic isotope effects for both phases (rapid burst phase and slow plateau phase) of the urease-catalyzed hydrolysis of hydroxyurea were measured at pH 6.0 and 25 degree [Celsius] to understand the observed nitrogen isotope effects for the ammonia leaving group and hydroxylamine leaving group. The observed similarity of the magnitude of the carbon isotope effects argues for formation of a common intermediate during both phases.

The discovery of a poly(ADP-ribose) glycohydrolase (PARG) regulatory segment far removed linearly from residues involved in catalysis is described. The presence of the regulated segment/N-terminal mitochondrial targeting sequence (REG/MTS) has increased the possibility that PARG activity is regulated by interactions of PARP-1 and -2 and other proteins at this site.

The metagenomic data mining and the genome sequencing of a marine bacterium, Zobellia galactanivorans, has helped in the identification of three new l-carrageenase genes belonging to the glycoside hydrolase family GH82. The results have shown that E310 has played a role in stabilizing the substrate intermediate conformation, while H281 is involved in substrate binding and is crucial for maintaining the protonation state of the catalytic proton donor E245.

The crystal structure of the urease maturation protein UreE from Helicobacter pylori is determined in its apo form, bound to [Cu.sup.2+] and [Ni.sup.2+]. The comparison of independent subunits from different crystal forms has shown changes in the relative arrangement of the N- and C-terminal domains in response to metal binding.

A new retention device was designed to perform transcriptomic analysis and explore the potential Aspergillus niger for product formation at very low specific growth rates. The results suggested that the improved retentostat method could be used in fundamental studies of differentiation and is applicable to filamentous fungi in general.

The biochemical properties as well as the nucleotide and amino acid sequences of arabinofuranosidase 3 (ABF3) from the soft rot fungus Penicillium purpurogenum are described. ABF3 is the first modular family 43 enzyme from a fungal source, which has both [alpha]-L-arabinofuranosidase and xylobiohydrolase functionalities.

Combination of site-directed mutagenesis and chemical modification are used to study the applicability of replacing putative catalytic base aspartyl residues, Aps392 and Asp216 with cysteinesulfinate, for the modulation of enzymatic properties. The obtained data demonstrated the potential use of combining site-directed mutagenesis with chemical modification as a feasible approach for the modulation of cellulases, and potentially other glycoside hydrolases, at low pH.

Bromoperoxidase A2 (BPO-A2) from Streptomyces aureofaciens was converted to a lipase by structure comparison with lipase A (LipA) from Bacillus subtilis to explore the plasticity of the [alpha]/[beta]-hydrolase fold family. The design strategy used in study provide insight into the structure-function relationship of the [alpha/[beta]-hydrolase fold enzymes and could be used as a valuable tool to construct variants with targeted catalytic properties using the [alpha]/[beta]-hydrolase fold.

The biochemical properties and crystal structures of a glycoside hydrolase (GH) families 5 and 26 mannanases and their contributions to substrate specificity on heterogeneous polymers are described. The results provide insight into the mechanism by which GH enzymes select for glucose or mannose at distal subsites which are critical to define their substrate specificicy and the biological rationale for the variable recognition of Man- and Glc-configured sugars by [beta]-mannanases.

A quorum-quenching lactonase from Mycobacterium avium subsp. paratuberculosis K-10 (GI: 41409766) as the initial template for in vitro evolution experiments was used to enhance the catalytic efficiency and increase the substrate range of the wild-type lactanose enzyme through a single point mutation on the loop at the C-terminal end of the eighth [beta]-strand. The studies established the evolutionary potential of the [([beta]/[alpha]).sub.8]-barrel fold and highlighted the possible application of quorum-quenching lactonases in the amidohydrolase superfamily as templates for engineering biomolecules of therapeutic use.

Several molecular dynamics simulations of the Trypanosoma cruzi trans-sialidase (TcTS) and Trypanosoma rangeli sialidase (TrSA) enzymes are conducted to explain the determinants of their structures and kinetic mechanism. TcTS is shown to exhibit a reduced solvent exposure, hence making it less efficient than the TrSA hydrolase.

A rapid and stereodivergent access to polyhydroxylated 10-azabicyclo[4.3.1]decanes as new calystegine analogues is achieved by a double benzotriazolyl/carbon nucleophile exchange followed by a ring-closing metathesis. The analysis of new compounds as glucocerebrosidase inhibitors is presented.

The allosteric properties of threonine deaminase from Bacillus subtilis (bsTD) are examined, which has only a single ACT-like domain. A possible evolutionary pathway is suggested to the more complex regulatory effects of valine on the activity of TDs of the long sequence variety.

The activity and crystal structure of bacterium Deinococcus radiodurans (DR_0079), the first nudix hydrolase which is observed to have a marked preference for cytosine ribonucleoside 5'-diphosphate (CDP) and cytosine ribonucleoside 5'-triphosphate (CTP) is reported. The protein is found to contain nine [beta]-strands, three [alpha]-helices, and two [3.sub.10]-helices organized into three subdomains, namely an N-terminal [beta]-sheet, a central nudix core, and a C-terminal helix-turn-helix motif.

The structure and characteristics of the ferrous human cystathionine [beta]-synthase (CBS) enzyme are studied. The results prove that mammalian CBS loses its activity during the enzyme assay due to a ligand switch process.

A study describes a novel mechanistic class of fatty acid amide hydrolase (FAAH) inhibitors with excellent selectivity. The analysis reveals that these FAAH inhibitors can easily be used as perfect analgesic and antidepressant agents.

A new functional proteomic strategy for developing inhibitors for uncharacterized serine hydrolases (SHs) is being described. The results show that carbamates can be easily used for the development of such inhibitors with excellent potency and selectivity.

The synthesis of adamantan-1-yl-methoxy-functionalized 1-deoxynojirimycin derivatives is demonstrated. The synthesis could be used to create a wide variety of lipophilic mono- and difunctionalized 1-deoxynojirimycin derivatives.

Four specific binuclear metallohydrolases, which are experimentally well-developed systems showing the geometric and electronic structures of active sites and transition states for these binuclear metal centers are highlighted. The discussion presented on binuclear metallohydrolases illustrates both mechanistic similarities and functional variations.

The structural properties of Bacillus pasteurii UreG (BpUreG) were studied in solution to obtain data that would clarify the molecular characteristics of UreG protein and elucidate the folded state of the protein. BpUreG contains a unique tryptophan residue (W192) which in the native state, is protected from the aqueous polar solvent while upon addition of denaturant a conformational change occurs that causes solvent exposure of the indole side chain.

The X-ray structure of the [NiFe] hydrogenase is determined in the Desulfovibrio gigas, Desulfovibrio fructosovorans, Desulfovibrio vulgaris, and Desulfomicrobium baculatum. It was found that the carbon monoxide acts as a reversible inhibitor of hydrogenases, and on the basis of spectroscopic and X-ray crystallographic study it was seen that the incoming CO was found to bound to Ni.

The catalytic contributions and mechanistic roles of the newly detected active site residues, as revealed by site-directed mutagenesis is described. The reason for the choice of histidine rather than glutamate as the general base is investigated by mutagenesis.

The structural and mechanistic diversity within the active site is described and correlated with the subsequent evolution of the reaction and substrate specificity in the assessment of the amidohydrlase superfamily. The amidohydrolase superfamily comprises a remarkable set of enzymes that catalyze the hydrolysis of a wide range of substrate bearing amide or ester functional groups at carbon and phosphorus centers.

The identification of four candidate residues for site-directed mutagenesis using the crystal structure of aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase [ATIC] is used for the study of cyclohydrolase activity of the bifunctional enzyme. The bifunctional enzyme ATIC is responsible for the catalysis of the last two steps in the de novo purine pathway.

A study of the inosine monophosphate cyclohydrolase [IMPCH] active site and the cyclohydrolase mechanism through a comparison of crystal structures of xanthosine 5'-monophosphate [XMP] inhibitor complexes of human aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase [ATIC] structure with the determined avian enzyme is presented. The structural comparison as well as comparative analysis with other Imp and XMP binding proteins gives insights into the cyclization reaction and of potential catalytic mechanism for IMPCH.

A study of celliobiohydrolase CbhA from Clostridium thermocellum, which is a large seven-modular enzyme with a catalytic module belonging to family 9, is presented. The exocellulolyitc activity of the enzyme is due to the altered loop region.