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L-Amino-acid ligases (LALs) are enzymes which catalyze the formation of dipeptides by linking two L-amino acids. Although many dipeptides are known and expected to have medical and nutritional benefits, their practical use has been limited owing to their low availability and high expense. LALs are potentially desirable tools for the efficient production of dipeptides; however, the molecular basis of substrate recognition by LAL has not yet been sufficiently elucidated for the design of ideal LALs for the desired dipeptides. This report presents the crystal structure of the LAL BL00235 derived fromBacillus licheniformisNBRC 12200 determined at 1.9 Å resolution using the multi-wavelength anomalous dispersion method. The overall structure of BL00235 is fairly similar to that of YwfE, the only LAL with a known structure, but the structure around the catalytic site contains some significant differences. Detailed structural comparison of BL00235 with YwfE sheds some light on the molecular basis of the substrate specificities.

Sirtuins are NAD(+)-dependent protein deacetylases regulating metabolism, stress responses, and aging processes. Mammalia have seven Sirtuin isoforms, Sirt1-7, which differ in their substrate specificities and subcellular localizations. The physiological functions of Sirtuins make them interesting therapeutic targets, which has stimulated extensive efforts on development of small molecule Sirtuin modulators. Yet, most Sirtuin inhibitors show limited potency and/or isoform specificity, and the mechanism of Sirtuin activation by small molecules remains obscure. Accumulating information on Sirtuin substrates, structures, and regulation mechanisms offer new opportunities for the challenging task to develop potent and specific small molecule modulators for mammalian Sirtuins for in vivo studies and therapeutic applications. We therefore recapitulate advances in structural and mechanistic studies on substrate recognition and deacetylation by Sirtuins, and in the characterization of compounds and molecular mechanisms regulating their activity. We then discuss challenges and opportunities from these findings for Sirtuin-targeted drug development efforts.

Because of their critical role in modulating cellular cyclic nucleotide levels, phosphodiesterases (PDEs) are involved in many disease-related signaling pathways. The PDE family is large and diverse, with members having different tissue distribution, sub-cellular localizations, and substrate specificities. Because of these characteristics, the PDEs represent a broad group of potential drug targets. Described in the present unit are the assay development and validation procedures needed to establish a high-throughput screening system for these important enzymes. The assays provide a structured approach for determining the kinetic parameters of related enzyme families to facilitate the characterization of PDE inhibitors.

Iron (Fe) is essential for plants but can be toxic if over-accumulated. Members of the yellow stripe-like (YSL) family of metal transporters play important roles in plant Fe homeostasis, and a great deal of evidence has been gathered over many years that indicates the importance of YSLs in the long distance transport of metals complexed with nicotianamine (NA). This review examines our current knowledge of YSLs, gleaned from both genetic and biochemical approaches. Many unanswered questions remain regarding the substrate specificities of these transporters, which seem to vary widely depending on the individual transporter. Data are also just beginning to become available regarding YSLs in the most basal clade, which may be responsible for intracellular transport of metal-NA complexes. Future research on YSL transporters should focus on utilizing the proven techniques of yeast complementation and Xenopus oocyte electrophysiology to examine the substrate specificity of YSLs in greater detail.

Plants are able to metabolize agrochemicals and other foreign compounds by a variety of mechanisms and with extraordinary species diversity. Minor structural alterations of these compounds can bring about dramatic and unpredictable changes in the routes of their metabolism. The enzymes responsible for this exist in multiple forms which renders prediction of herbicide metabolism and, therefore, of selectivity, difficult. In some notable instances, pesticides are activated by plant metabolism. In the main, however, mechanisms such as hydroxylation, dealkylation and glutathione conjugation bring about detoxification and form the basis of herbicide selectivity. The properties of oxygenating and conjugating enzymes in plants are highlighted, with emphasis on the evident narrow substrate specificities, species differences and physiological roles. The molecular cloning of the genes specifying these enzymes will permit a much better definition of these mechanisms and will illuminate the natural roles of the enzymes involved. The prospects for utilizing recombinant enzymes as tools for the rational design of new selective herbicides are discussed. Herbicide safeners can protect certain crops from herbicide injury by promoting herbicide metabolism. The precise mechanisms of safener action and the reasons for their specificity are attracting much interest but are at present obscure. Natural variation in detoxification abilities of weed populations has allowed the field selection of some biotypes resistant to repeatedly used herbicides. By analogy, the introduction of microbial detoxification genes into major crops through genetic transformation has created new herbicideresistant crops which will enhance the flexibility of herbicide usage.

The glutathione-S-transferase (GST) proteins represent an extended family involved in detoxification processes. They are divided into various classes with high diversity in various organisms. The Ure2p class is especially expanded in saprophytic fungi compared to other fungi. This class is subdivided into two subclasses named Ure2pA and Ure2pB, which have rapidly diversified among fungal phyla. We have focused our analysis on Basidiomycetes and used Phanerochaete chrysosporium as a model to correlate the sequence diversity with the functional diversity of these glutathione transferases. The results show that among the nine isoforms found in P. chrysosporium, two belonging to Ure2pA subclass are exclusively expressed at the transcriptional level in presence of polycyclic aromatic compounds. Moreover, we have highlighted differential catalytic activities and substrate specificities between Ure2pA and Ure2pB isoforms. This diversity of sequence and function suggests that fungal Ure2p sequences have evolved rapidly in response to environmental constraints.

Hydantoinases/dihydropyrimidinases are important biotechnological enzymes involved in the production of α- and β-amino acids. Their isolation from new sources with different substrate specificities, improved activity, enantioselectivity, or higher stability continues to be of great industrial interest. Here, we provide a detailed description of how to produce high quantities of the recombinant hydantoinase/dihydropyrimidinase enzyme from Sinorhizobium meliloti CECT4114 (SmeDhp). Several techniques are combined to obtain this goal, from cloning to activity measurement by HPLC.

Multi-copper oxidases are a large family of enzymes prevalent in all three domains of life. They couple the one-electron oxidation of substrate to the four-electron reduction of dioxygen to water and feature at least four Cu atoms, traditionally divided into three sites: T1, T2, and (binuclear) T3. The T1 site catalyzes substrate oxidation while a trinuclear cluster (comprising combined T2 and T3 centres) catalyzes the reduction of dioxygen. Substrate oxidation at the T1 Cu site occurs via an outer-sphere mechanism and consequently substrate specificities are determined primarily by the nature of a substrate docking/oxidation (SDO) site associated with the T1 Cu centre. Many of these enzymes ‘moonlight’, i.e. display broad specificities towards many different substrates and may have multiple cellular functions. A sub-set are robust catalysts for the oxidation of low-valent transition metal ions such as FeII, CuI, and MnII and are termed ‘metallo-oxidases’. They play essential roles in nutrient metal uptake and homeostasis, with the ferroxidase ceruloplasmin being a prominent member. Their SDO sites are tailored to facilitate specific binding and facile oxidation of these low-valent metal ions and this is the focus of this review.

Directed evolution is widely used to improve enzymes, particularly for industrial biocatalytic processes. Molecular biology advances present many new strategies for directed evolution. Commonly used techniques have led to many successful examples of enzyme improvement, yet there is still a need to improve both the efficiency and capability of directed evolution. Recent strategies aimed at making directed evolution faster and more efficient take better advantage of available structural and sequence information. The underlying principles that lead to early dead-ends for directed evolution experiments are also discussed along with recent strategies designed to by-pass them. Several emerging methods for creating novel enzymes are also discussed including examples of catalytic activity for which there is no precedent in nature. Finally, the combined use of several strategies is likely to be required in practice to improve multiple target properties of an enzyme, as successfully shown by a recent industrial example.

We have previously reported the structures of the native holo and substrate-bound forms of ll -diaminopimelate aminotransferase from Arabidopsis thaliana (AtDAP-AT). Here, we report the crystal and molecular structures of the ll -diaminopimelate aminotransferase from Chlamydia trachomatis (CtDAP-AT) in the apo-form and the pyridoxal-5′-phosphate-bound form. The molecular structure of CtDAP-AT shows that its overall fold is essentially identical with that of AtDAP-AT except that CtDAP-AT adopts an “open” conformation as opposed to the “closed” conformation of AtDAP-AT. Although AtDAP-AT and CtDAP-AT are approximately 40% identical in their primary sequence, they have major differences in their substrate specificities; AtDAP-AT is highly specific for LL-DAP, whereas CtDAP-AT accepts a wider range of substrates. Since all of the residues involved in substrate recognition are highly conserved between AtDAP-AT and CtDAP-AT, we propose that differences in flexibility of the loops lining the active-site region between the two enzymes likely account for the differences in substrate specificity.

An enzyme's substrate specificity is one of its most important characteristics. The quantitative comparison of broad-specificity enzymes requires the selection of a homogenous set of substrates for experimental testing, determination of substrate-specificity data and analysis using multivariate statistics. We describe a systematic analysis of the substrate specificities of nine wild-type and four engineered haloalkane dehalogenases. The enzymes were characterized experimentally using a set of 30 substrates selected using statistical experimental design from a set of nearly 200 halogenated compounds. Analysis of the activity data showed that the most universally useful substrates in the assessment of haloalkane dehalogenase activity are 1-bromobutane, 1-iodopropane, 1-iodobutane, 1,2-dibromoethane and 4-bromobutanenitrile. Functional relationships among the enzymes were explored using principal component analysis. Analysis of the untransformed specific activity data revealed that the overall activity of wild-type haloalkane dehalogenases decreases in the following order: LinB~DbjA>DhlA~DhaA~DbeA~DmbA>DatA~DmbC~DrbA. After transforming the data, we were able to classify haloalkane dehalogenases into four SSGs (substrate-specificity groups). These functional groups are clearly distinct from the evolutionary subfamilies, suggesting that phylogenetic analysis cannot be used to predict the substrate specificity of individual haloalkane dehalogenases. Structural and functional comparisons of wild-type and mutant enzymes revealed that the architecture of the active site and the main access tunnel significantly influences the substrate specificity of these enzymes, but is not its only determinant. The identification of other structural determinants of the substrate specificity remains a challenge for further research on haloalkane dehalogenases.

A bacterial flavin-containing monooxygenase (FMO), fused to phosphite dehydrogenase, has been used to explore its biocatalytic potential. The bifunctional biocatalyst could be expressed in high amounts in Escherichia coli and was able to oxidize indole and indole derivatives into a variety of indigo compounds. The monooxygenase also performs the sulfoxidation of a wide range of prochiral sulfides, showing moderate to good enantioselectivities in forming chiral sulfoxides.

1. Monoclonal antibodies were raised against squid hepatopancreas organophosphorous acid (OPA) anhydrolase (EC 3.1.8.2) and were used to study structural similarities with OPA anhydrolases isolated from different sources. 2. Common epitopes were identified in OPA anhydrolases with diverse origins, and with different substrate specificities. 3. Epitopes unique to the squid hepatopancreas OPA anhydrolase were identified; optic ganglion and hepatopancreas contain different enzymes which can be distinguished by their epitopes.

Chloroaromatic compounds are widely used and may cause serious pollution. Aerobic biodegradation of some chloroaromatics by microorganisms has been studied extensively and an increasing number of oxygenase genes responsible for the degradation of aromatic compounds, including chloroaromatics, are being characterized. Chloroaromatic-degrading oxygenases show limited divergence. It seems that chloroaromatic oxygenases have evolved from a limited number of ancestral oxygenases specific for non-substituted substrates by broadening their substrate ranges or altering their substrate specificities.

Activities and substrate specificities of crude enzymes of dimethylthetin-homocysteine (DMT-Hcys) and betaine-homocysteine (Bet-Hcys) S-methyltransferases from livers of fish (12 species), cattle, rat, and chicken were examined. Specific activities of DMT-Hcys enzyme were higher than those of Bet-Hcys enzyme in red sea bream and yellowtail, but vice versa in rainbow trout, flounder, and rat. In carp, cattle, and chicken, specific activities of both enzymes were almost the same. Substrate specificities of the DMT-Hcys enzymes from the liver of red sea bream were examined on a number of structurally similar compounds to DMT. Of all the test compounds, dimethylpropiothetin proved to be the most efficient substrate for the enzyme.

Protein microarray technology has emerged as a powerful tool for comparing binding interactions, expression level, substrate specificities, and posttranslational modifications (PTMs) of different proteins in a parallel and high-throughput manner. The ability to immobilize proteins to a solid surface and register the specific address of each protein has bridged major limitations for investigating the proteome in biological samples, namely, the wide dynamic range of protein concentrations and the perturbation of the physical and chemical properties of proteins by their modification. Recent advances introduced the use of functional mammalian cell extracts to assay PTMs under different cellular conditions. This assay offers a new approach for performing large-scale complex biochemical analysis of protein modifications. Here, we review studies of PTM profiling using protein microarrays and discuss the limitations and potential applications of the system. We believe that the information generated from such proteomic studies may be of significant value in our elucidation of the molecular mechanisms that govern human physiology.

Vascular adhesion protein-1 (VAP-1) belongs to the semicarbazide-sensitive amine oxidases (SSAOs) that convert amines into aldehydes. SSAOs are distinct from the mammalian monoamine oxidases (MAOs), but their substrate specificities are partly overlapping. VAP-1 has been proposed as a target for anti-inflammatory drug therapy because of its role in leukocyte adhesion to endothelium. Here, we describe the synthesis and in vitro activities of novel series of VAP-1 selective inhibitors. In addition, the molecular dynamics simulations performed for VAP-1 reveal that the movements of Met211, Ser496, and especially Leu469 can enlarge the ligand-binding pocket, allowing larger ligands than those seen in the crystal structures to bind. Combining the data from molecular dynamics simulations, docking, and in vitro measurements, the three-dimensional quantitative structure-activity relationship (3D QSAR) models for VAP-1 (q(2)(LOO): 0.636; r(2): 0.828) and MAOs (q(2)(LOO): 0.749, r(2): 0.840) were built and employed in the development of selective VAP-1 inhibitors.

The CH/π-interaction is a weak hydrogen bond occurring between CH groups and π-electron systems. Evidence for such a weak attractive molecular force has been presented. These include data from IR, NMR, CD spectroscopies, and X-ray crystallography. The conclusion has been supported by ab initio MO calculations. An important point is that CH- and π-groups are arranged generally in chemical structures and have many chance to interact each other. Unlike ordinary hydrogen bondings, the CH/π interaction may occur in protic media as well as in nonpolar atmosphere. Consequences in supramolecular chemistry, selectivities in chemical reactions, as well as the substrate specificities of proteins were discussed in the light of the CH/π-interaction hypothesis.

Xylanases are of widespread importance in several food and non-food biotechnological applications. They degrade heteroxylans, a structurally heterogeneous group of plant cell wall polysaccharides, and other important components in various industrial processes. Because of the highly complex structures of heteroxylans, efficient utilization of xylanases in these processes requires an in-depth knowledge of their substrate specificity. A significant number of studies on the three-dimensional structures of xylanases from different glycoside hydrolase (GH) families in complex with the substrate provided insight into the different mechanisms and strategies by which xylanases bind and hydrolyze structurally different heteroxylans and xylo-oligosaccharides (XOS). Combined with reports on the hydrolytic activities of xylanases on decorated XOS and heteroxylans, major advances have been made in our understanding of the link between the three-dimensional structures and the substrate specificities of these enzymes. In this review, authors gave a concise overview of the structure-function relationship of xylanases from GH5, 8, 10, and 11. The structural basis for inter- and intrafamily variation in xylanase substrate specificity was discussed as are the implications for heteroxylan degradation.

Directed enzyme evolution has proven to be a powerful tool for improving a range of properties of enzymes through consecutive rounds of diversification and selection. However, its success depends heavily on the efficiency of the screening strategy employed. Fluorescence-activated cell sorting (FACS) has recently emerged as a powerful tool for screening enzyme libraries due to its high sensitivity and its ability to analyze as many as 10(8) mutants per day. Applications of FACS screening have allowed the isolation of enzyme variants with significantly improved activities, altered substrate specificities, or even novel functions. This review discusses FACS-based screening for enzymatic activity and its potential application for the directed evolution of enzymes, ribozymes, and catalytic antibodies.

Building Units of Oligosaccharides, CIV. — Synthesis of Branched Tetrasaccharide and Pentasaccharide Structures of N-Glycoproteins Methylated at 4′-OH of the Branching Unit The tetrasaccharide α-D-Manp-(13)[α-D-Manp-(16)]-4-O-methyl-β-D-Manp-(14)-D-GlcNAc (15) and the pentasaccharide β-D-GlcpNAc-(12)-α-D-Manp- (3)[α-D-Manp-(6)]-4-O-methyl-β-D-Manp- (4)-D-GlcNAc (23) were synthesized by adding the respective functionalized building blocks. The compounds are useful for studies of the substrate specificities of GlcNAc transferases I and II in the biosynthesis of N-linked oligosaccharides, respectively. In addition we developed an effective synthesis for the β-glycosidically linked building block β-D-Manp-(4)-α-D-GlcpNAc. The trichloroacetimidate method was particularly successful for synthesizing these glycosidic linkages.

The bakers’ yeast reduction of a series of 2-oxoalkyl arenecarboxylates [R(C=O)CH2O(C=O)C6H4-p-X] (1a–f) (R = CH3 to n-C6H13; X = H) and the phenyl-modified derivatives (1g–l) (R = n-C5H11, X = OH, CH3, F, Cl, Br, or I) as well as 1-chloro-2-alkanones R(C=O)CH2Cl (6a–f) (R = CH3 to n-C6H13) were systematically investigated. The substrate specificities, configuration and %ee of the reduction products were found to be highly dependent on the length of the alkyl group (R) and the α substituent. Thus, the benzoates 1a–f gave optically active 2-hydroxyalkyl benzoates (2a–f) (R, configuration, %ee) (a: CH3, S, 99; b: C2H5, S, 98; c: C3H7, S, 26; d: n-C4H9, R, 55; e: n-C5H11, S, 15; f: n-C6H13, S, 63) in 11–91% yields. Among the modification experiments of the phenyl group, 1g–l, the p-iodo substituent markedly increased the ee from 15 to 71%, although the yield was rather lowered (22% yield). The reduction of α-chloro ketones 6a–f also gave optically active 1-chloro-2-alkanols (7a–f) [(R, configuration, %ee) (a...

Lipoxygenases were highly purified from soybean seed, wheat seed and cucumber cotyledons. Substrate specificities of these lipoxygenases were studied by using an entire series of (ω6Z ,ω9Z )-C13~C24-dienoic acids as synthetic substrate analogues. Soybean lipoxygenase-1 and cucumber lipoxygenase showed broad specificities for these substrates while wheat lipoxygenase showed narrow specificities. Position of dioxygenation to each substrate was analyzed by high performance liquid chromatography. With soybean lipoxygenase-1 elongation of the distance between the terminal carboxyl group and the site of hydrogen removal in a substrate decreased the positional specificity of dioxygenation, while, with cucumber lipoxygenase, shortening the distance decreased the specificity. It was suggested that cucumber lipoxygenase and soybean lipoxygenase-1 recognized the terminal carboxyl group of a substrate to arrange it only in one orientation at the reaction center. In case of wheat lipoxygenase, recognition of the carboxyl group was thought to have crucial and essential role to secure the activity.

Cytochrome P450 enzymes (P450s) are able to regioselectively and stereoselectively introduce oxygen into organic compounds under mild reaction conditions. These monooxygenases in particular easily catalyze the insertion of oxygen into less reactive carbon-hydrogen bonds. Hence, P450s are of considerable interest as oxidation biocatalysts. To date, although several P450s have been discovered through screening of microorganisms and have been further genetically engineered, the substrate range of these biocatalysts is still limited to fulfill the requirements for a large number of oxidation processes. On the other hand, the recent rapid expansion in the number of reported microbial genome sequences has revealed the presence of an unexpectedly vast number of P450 genes. This large pool of naturally evolved P450s has attracted much attention as a resource for new oxidation biocatalysts. In this review, we focus on aspects of the genome mining approach that are relevant for the discovery of novel P450 biocatalysts. This approach opens up possibilities for exploitation of the catalytic potential of P450s for the preparation of a large choice of oxidation biocatalysts with a variety of substrate specificities.

The carbon–fluorine bond is one of the strongest in nature, and the increasing use of organofluorine compounds in agriculture, human and veterinary medicine, and industry has raised concerns about their fate in the environment. Microorganisms can degrade organofluorine compounds, either via specific enzymatic hydrolysis of the C–F bond, or through transformation by catabolic enzymes with broad substrate specificities. Here our current understanding of organofluorine catabolism in microorganisms is summarised.

A TP-binding cassette (ABC) multidrug transporters are cellular efflux pumps with broad and often widely overlapping substrate specificities. They can have a major impact on the pharmacokinetics and hence overall pharmacological behavior of many drugs. To study their separate roles and functional overlap, or complementarity, a collection of mice deficient in two or more ABC transporters has been generated. This review discusses recent findings obtained with these models, focusing on pharmacokinetic studies with a number of clinically relevant drugs. In addition, the characterization of these mice and some physiological aspects of ABC multidrug transporters are addressed.

In recent years, endogenous and xenobiotic metabolism has been one of the cutting edge topics of our journal (Hengstler and Bolt 2008; Bolt and Hengstler 2007, 2008a, b). Because of the dynamic progress in the field of drug metabolizing enzymes, we have initiated a new series of review articles that focus on nomenclature, substrate specificities, genetic organization, polymorphisms, clinical relevance and role in carcinogenesis (Pelkonen et al. 2008; Lankisch et al. 2008; Strassburg et al. 2008; Decker et al. 2009; Adam and Laufs 2008; Macdonald and Gledhill 2007). In order to give our readers a better overview over the numerous articles published in the field of drug metabolism in our journal during the past 2 years, we summarize their key messages and link them to the respective articles (Table 1).

The existence of carrier proteins located in the basolateral and apical membranes of hepatocytes, cholangiocytes and epithelial cells of the ileal mucosa, together with their more or less broad substrate specificities -implying their ability to transport many different drugs, including anticancer drugs- has important pharmacological repercussions. These vary from the existence of interactions of drugs with endogenous and xenobiotic substances to the possibility of using these transporters in the targeting of drug delivery systems, which can be useful either to direct anticancer drugs towards tumors located in the hepatobiliary system or to facilitate their hepatobiliary excretion. This justifies the growing interest in bile acid derivatives as targeted pharmacological tools, in general, and in anticancer chemotherapy, in particular. Moreover, interactions of antitumor drugs with hepatobiliary transporters may account for the appearance of toxic side effects associated with the use of these drugs. The present review covers these aspects of the pharmacology and toxicology of hepatobiliary transport systems in relation to anticancer drugs.

We have succeeded in the purification and cDNA cloning of several enzymes involved in the biosynthesis of biologically important N-glycans of glycoproteins. These enzymes are responsible for the formation of branching structures of the core of N-glycans. To detect the activities with ease and sensitivity, we developed assay methods for these glycosyltransferases including N-acetylglucosaminyltransferases (GnTs) III, IV, V, VI, and IX using fluorescence-labeled oligosaccharides as acceptor substrates. The branching structures of N-glycans such as β1,6 GlcNAc branching and bisecting GlcNAc are formed by the actions of GnT-V and GnT-III, respectively. It has been reported that these N-glycans may play important roles in cell growth, cancer invasion, and metastasis. The assay methods for glycosyltransferases, including these enzymes involved in the branching formation of the core structure of N-glycan, are described (Fig. 1). The procedure described here is based on the methodology originally developed by Hase et al. (1978) and reported by our group earlier (Taniguchi et al. 1989). Each enzymatic product can be separated from the reaction mixture using reversed-phase HPLC. These methods allowed us to assay the activities with high sensitivity and substrate specificities of these glycosyltransferases.

Many studies using monoclonal antibodies have demonstrated that fucosylated carbohydrate chains are involved in various physiological functions including development, neuronal activity, and cancerous transformation. The genes encoding fucosyltransferases which synthesize fucosylated carbohydrate antigens have been cloned, and their function has been gradually elucidated by analyses of their substrate specificities and biological functions (Taniguchi et al. 2002).

In the synthesis of glycans, it is best to apply organic chemistry if we require a large amount of single structure, while an enzymatic synthesis using various glycosyltransferases with different substrate specificities is a suitable method when we need a variety of glycans in small quantity. However, commercially available glycosyltransferases are limited in variety which then restricts the types of synthesizable glycans. The structural and functional analyses of glycans involve the syntheses of various glycan structures, hence it is vital to have a variety of glycosyltransferases.

The recent progress in knowledge on biochemical properties and functions of phospholipases A(2) in plants paved the way for approving the suitability of these enzymes for commercial use now. The secreted phospholipases A(2), representing one type of phospholipases A(2) occurring in plants, show distinct differences in substrate specificities with respect to headgroup and acyl chains of the glycerophospholipids in comparison to their counterparts from animal sources. The other type of phospholipases A(2) in plants, the patatin-related phospholipases A(2), is characterized by broad substrate specificity. Accordingly, the unique properties of the plant enzymes open new horizons to engineered biocatalysts with improved performance, e.g., for vegetable oil refinement by degumming and for targeted modification of phospholipids.

Two glycoside hydrolase (GH) families were analyzed to detect the presence of functional divergence using the program DIVERGE. These two families, GH7 and GH16, each contain members related by amino acid sequence similarity, retaining hydrolytic mechanisms, and catalytic residue identity. GH7 and GH16 comprise GH Clan B, with a shared β-jelly roll topology and mechanism. GH7 contains fungal cellobiohydrolases and endoglucanases and is divided into five main subfamilies, four of the former and one of the latter. Cluster comparisons between three of the cellobiohydrolase subfamilies and the endoglucanase subfamily identified specific amino acid residues that play a role in the functional divergence between the two enzyme types. GH16 contains subfamilies of bacterial agarases, xyloglucosyl transferases, 1,3-β-D-glucanases, lichenases, and other enzymes with various substrate specificities and product profiles. Four cluster comparisons between these four main subfamilies again have identified amino acid residues involved in functional divergence between the subfamilies. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 478–495, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

The crystal structure of a putative dipeptidase (Phdpd) fromPyrococcus horikoshiiOT3 was solved using X-ray data at 2.4 Å resolution. The protein is folded into two distinct entities. The N-terminal domain consists of the general topology of theα/βfold, and the C-terminal domain consists of five long mixed strands, four helices, and two310helices. The structure ofPhdpd is quite similar to reported structures of prolidases fromP. furiosus(Zn-Pfprol) andP. horikoshii(Zn-Phdpd), where Zn ions are observed in the active site resulting in an inactive form. However,Phdpd did not contain metals in the crystal structure and showed prolidase activity in the absence of additional Co ions, whereas the specific activities increased by 5 times in the presence of a sufficient concentration (1.2 mM) of Co ions. The substrate specificities (X-Pro) ofPhdpd were broad compared with those of Zn-Phdpd in the presence of Co ions, whose relative activities are 10% or less for substrates other than Met-Pro, which is the most favorable substrate. The binding constants of Zn-Phdpd with three metals (Zn, Co, and Mn) were higher than those ofPhdpd and that with Zn was higher by greater than 2 orders, which were determined by DSC experiments. From the structural comparison of both forms and the above experimental results, it could be elucidated why the protein withZn2+ions is inactive.

Directed evolution can generate a remarkable range of new enzyme properties. Alternate substrate specificities and reaction selectivities are readily accessible in enzymes from families that are naturally functionally diverse. Activities on new substrates can be obtained by improving variants with broadened specificities or by step-wise evolution through a sequence of more and more challenging substrates. Evolution of highly specific enzymes has been demonstrated, even with positive selection alone. It is apparent that many solutions exist for any given problem, and there are often many paths that lead uphill, one step at a time.

Protein phosphorylation is a crucial regulatory mechanism in various organisms. With recent improvements in mass spectrometry, phosphorylation site data are rapidly accumulating. Despite this wealth of data, computational prediction of phosphorylation sites remains a challenging task. This is particularly true in plants, due to the limited information on substrate specificities of protein kinases in plants and the fact that current phosphorylation prediction tools are trained with kinase-specific phosphorylation data from non-plant organisms. In this paper, we proposed a new machine learning approach for phosphorylation site prediction. We incorporate protein sequence information and protein disordered regions, and integrate machine learning techniques of k-nearest neighbor and support vector machine for predicting phosphorylation sites. Test results on the PhosPhAt dataset of phosphoserines in Arabidopsis and the TAIR7 non-redundant protein database show good performance of our proposed phosphorylation site prediction method.

Drug transporters expressed on the hepatocyte membrane play an important role in hepatic drug disposition. In the last two decades, systematic research has resulted in a better understanding of the diversity, expression and substrate specificities of drug transporters in the liver. Here we review recent studies on the role of transporters in drug-drug interactions and disease states such as cirrhosis. We conclude the review by considering techniques and model systems used to study hepatic transporters, including the latest technological developments such as multiphoton microscopy. Refereed/Peer-reviewed

New substrate specificities can be introduced into existing enzymes for the purpose of making them more suitable for the chemoenzymic synthesis of single compound drugs and other chiral compounds. The most productive route used in the past year has involved the utilization of the catalytic and substrate-binding properties from homologous enzymes found in nature, one example being the broadening of the substrate specificity of yeast alcohol dehydrogenase. Other highlights include the creation of thermostable dehydrogenases that will interconvert NADPH and NADH, and the design of mutant enzymes with improved catalytic rates compared with their wild-type counterparts.

The first structure of a chalcone synthase has been determined. Enzymes in this family have broad substrate specificities and participate in the biosynthesis of many secondary plant products, including compounds that provide defense against pathogens and precursors to the bitter acids in hops that flavor beer.

Ribonucleases (RNases) with different sequence or structural specificities are used for a variety of analytical purposes, including RNA sequencing, mapping, and quantitation. The development of RNase protection assays, structural determination assays, and the production of small interfering RNAs (siRNA) employed in RNA interference (RNAi) experiments has depended on the unique substrate specificities of commercially available RNases, including RNases A, I, T1, V1, HI, III, and Dicer. One very common application for high purity RNase A is also presented in this unit and involves hydrolyzing RNA that contaminates DNA preparations. RNase HII and the placental RNase inhibitor are also discussed. Curr. Protoc. Mol. Biol. 84:3.13.1-3.13.8. © 2008 by John Wiley & Sons, Inc. Keywords: RNase; RNase protection assay; RNA footprinting; RNAi; siRNA

Among the major CYP subfamilies participating in xenobiotic metabolism, the CYP2A subfamily has received relatively little attention. This is due to the facts that the human CYP2A enzymes have very restricted substrate specificities and they are minor hepatic forms in quantitative terms. However, in...

An intracellular nitrilase was purified from a Fusarium solani O1 culture, in which the enzyme (up to 3000 U L−1) was induced by 2-cyanopyridine. SDS-PAGE revealed one major band corresponding to a molecular weight of approximately 40 kDa. Peptide mass fingerprinting suggested a high similarity of the protein with the putative nitrilase from Gibberella moniliformis. Electron microscopy revealed that the enzyme molecules associated into extended rods. The enzyme showed high specific activities towards benzonitrile (156 U mg−1) and 4-cyanopyridine (203 U mg−1). Other aromatic nitriles (3-chlorobenzonitrile, 3-hydroxybenzonitrile) also served as good substrates for the enzyme. The rates of hydrolysis of aliphatic nitriles (methacrylonitrile, propionitrile, butyronitrile, valeronitrile) were 14–26% of that of benzonitrile. The nitrilase was active within pH 5–10 and at up to 50 °C with optima at pH 8.0 and 40–45 °C. Its activity was strongly inhibited by Hg2+ and Ag+ ions. More than half of the enzyme activity was preserved at up to 50% of n-hexane or n-heptane or at up to 15% of xylene or ethanol. Operational stability of the enzyme was examined by the conversion of 45 mM 4-cyanopyridine in a continuous and stirred ultrafiltration-membrane reactor. The nitrilase half-life was 277 and 10.5 h at 35 and 45 °C, respectively.

In addition to synthetic and semisynthetic methods, new anthracycline structures have been generatedby biosynthetic methods: genetic engineering of Streptomyces productionstrains, bioconversion and chemoenzymatic synthesis. In this review, we discuss the set of moleculespotentially producible by biosynthetic methods and which structures have so far been realized. Biosyntheticvariation in the anthracycline molecule manifests itself either as structure changes in the tetracyclicaglycone, or as variation in glycosylation. Understanding the biosynthetic sequence and knowledgeof the substrate specificities of the enzymes participating in it enable rational generation of newanthracycline diversity. Future possibilities include protein engineering of the biosynthetic enzymesto improve the production of new structural combinations.

Two methods were investigated for the enzymic preparation of trehalose-containing trisaccharides. In the first, a solution of saccharides is circulated through an immobilized-glycosidase column and an activated-carbon column connected in series. In the second, two enzymes having different substrate specificities are sequentially used for condensation and subsequent specific hydrolysis. Thus, 3- O -β- d -, 4- O -β- d -, and 6- O -β- d -galactopyranosyl- a , a -trehalose; 4- O - a - d - and 6- O - a - d -glucopyranosyl- a , a -trehalose; and 4- O -β- d - and 6- O -β- d -glucopyranosyl- a , a -trehalose were synthesized stereo- and regio-selectively.

Currently, the major recognized biochemical functions of members of the large superfamily of P450 hemoproteins (referred to commonly as the cytochromes P450) include catalyses of the monooxygenations of a wide variety of endogenous and exogenous lipophilic chemicals. Substrates that have attracted the greatest attention thus far are steroids, fatty acids, eicosanoids, retinoids, other endogenous lipids, therapeutic agents, pesticides/herbicides, chemical carcinogens, industrial chemicals and other environmental contaminants and toxic xenobiotic organics of low molecular weight. Commonly, monooxygenation of such substrates results in the generation of metabolites capable of producing biological effects that are profoundly different (qualitatively as well as quantitatively) from those elicitable by the parent chemical per se. P450XIX-dependent conversion of testosterone to estradiol-17β provides a dramatic example. Thus, these hemoproteins serve as extremely important but, as yet, largely unpredictable regulators of the biological effects producible by endobiotics as well as by xenobiotics. Current focus is on the identification and acquisition of sequence information on hereto unidentified and/or uncharacterized P450 isoforms and ascertainment of the specific functions of specific, individual isoforms. The regulation of quantities and activities of such isoforms in specific species/tissues, understandably, is also of great current interest. This interest has been further intensified by recent results indicating that substrate specificity associated with one P450 may not be the same as the corresponding isoform derived from a different animal species. Recent technological advances promise to greatly hasten the acquisition of knowledge concerning the functions of the these important hemoproteins.