Identification of Structure-Stabilizing Interactions in Enzymes: A Novel Mechanism to Impact Enzyme Activity

Cruzain, a cysteine protease in the cathepsin family, is pivotal to the life-cycle of Trypanosoma cruzi, the etiological agent in Chagas disease. Current inhibitors of cruzain suffer from drawbacks involving gastrointestinal and neurological side effects and as a result have spurred the search for alternative anti-trypanocidals. Through sequence alignment studies and intra-residue interaction analysis of the pro-protein of cruzain (pro-cruzain), we have identified a host of non-active site residues that are conserved among the cathepsins. We hypothesize that these conserved amino acids play a critical role in structure-stabilizing interactions among the cathepsins and are therefore crucial for eventually gaining protease activity. As predicted, mutation of selected conserved non-active site amino-acid candidates in cruzain resulted in a compromised structural stability and a corresponding loss in enzymatic activity relative to wild-type enzyme. By advancing the discovery of novel, non-active-site-based targets to arrest enzymatic activity our results potentially open the field of alternative inhibitor design. The advantages of defining such a non-active-site inhibitor design space is discussed.     

Exploring the Enantioselective Mechanism of Halohydrin Dehalogenase from Agrobacterium radiobacter AD1 by Iterative Saturation Mutagenesis

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential in producing valuable chiral epoxides and β-substituted alcohols. The wild-type (WT) enzyme displays a high R-enantiopreference toward most aromatic substrates, whereas no S-selective HheC has been reported to date. To obtain more enantioselective enzymes, seven noncatalytic active-site residues were subjected to iterative saturation mutagenesis (ISM). After two rounds of screening aspects of both activity and enantioselectivity (E), three outstanding mutants (Thr134Val/Leu142Met, Leu142Phe/Asn176His, and Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutants) with divergent enantioselectivity were obtained. The two double mutants displayed approximately 2-fold improvement in R-enantioselectivity toward 2-chloro-1-phenylethanol (2-CPE) without a significant loss of enzyme activity compared with the WT enzyme. Strikingly, the Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutant showed an inverted enantioselectivity (from an E_R of 65 [WT] to an E_S of 101) and approximately 100-fold-enhanced catalytic efficiency toward (S)-2-CPE. Molecular dynamic simulation and docking analysis revealed that the phenyl side chain of (S)-2-CPE bound at a different location than that of its R-counterpart; those mutations generated extra connections for the binding of the favored enantiomer, while the eliminated connections reduced binding of the nonfavored enantiomer, all of which could contribute to the observed inverted enantiopreference.     

Imaging Enzymes at Work: Metabolic Mapping by Enzyme Histochemistry

For the understanding of functions of proteins in biological and pathological processes, reporter molecules such as fluorescent proteins have become indispensable tools for visualizing the location of these proteins in intact animals, tissues, and cells. For enzymes, imaging their activity also provides information on their function or functions, which does not necessarily correlate with their location. Metabolic mapping enables imaging of activity of enzymes. The enzyme under study forms a reaction product that is fluorescent or colored by conversion of either a fluorogenic or chromogenic substrate or a fluorescent substrate with different spectral characteristics. Most chromogenic staining methods were developed in the latter half of the twentieth century but still find new applications in modern cell biology and pathology. Fluorescence methods have rapidly evolved during the last decade. This review critically evaluates the methods that are available at present for metabolic mapping in living animals, unfixed cryostat sections of tissues, and living cells, and refers to protocols of the methods of choice. (J Histochem Cytochem 58:481–497, 2010)     

Novel Subtype of Peroxisomal Acyl-CoA Oxidase Deficiency and Bifunctional Enzyme Deficiency with Detectable Enzyme Protein: Identification by Means of Complementation Analysis

We describe four infants with a novel subtype of an isolated deficiency of one of the peroxisomal β-oxidation enzymes with detectable enzyme protein. The patients showed characteristic clinical and biochemical abnormalities, including hypotonia, psychomotor retardation, hepatomegaly, typical facial appearance, accumulation of very-long-chain fatty acids, and decreased lignoceric acid oxidation. However, β-oxidation enzyme proteins were detected by immunoblot analyses, and large peroxisomes were identified by immunofluorescence staining. In order to identify the underlying defect in these patients, complementation analysis was introduced using fibroblasts from these patients and patients with an established deficiency of either acyl-CoA oxidase or bifunctional enzyme, as identified by immunoblotting. In the complementing combinations, fused cells showed increased lignoceric acid oxidation, resistance against 1-pyrene dodecanoic acid/UV selection, and normalization of the size and the distribution of peroxisomes. The results indicate that two patients with a more severe clinical course were suffering from bifunctional enzyme deficiency and that the other two infants, who were siblings and had a less severe clinical presentation, were the first patients with acyl-CoA oxidase deficiency with detectable enzyme protein.     

A Novel Alkaliphilic Bacillus Esterase Belongs to the 13th Bacterial Lipolytic Enzyme Family

Background

Microbial derived lipolytic hydrolysts are an important class of biocatalysts because of their huge abundance and ability to display bioactivities under extreme conditions. In spite of recent advances, our understanding of these enzymes remains rudimentary. The aim of our research is to advance our understanding by seeking for more unusual lipid hydrolysts and revealing their molecular structure and bioactivities.

Methodology/Principal Findings

Bacillus. pseudofirmus OF4 is an extreme alkaliphile with tolerance of pH up to 11. In this work we successfully undertook a heterologous expression of a gene estof4 from the alkaliphilic B. pseudofirmus sp OF4. The recombinant protein called EstOF4 was purified into a homologous product by Ni-NTA affinity and gel filtration. The purified EstOF4 was active as dimer with the molecular weight of 64 KDa. It hydrolyzed a wide range of substrates including p-nitrophenyl esters (C2–C12) and triglycerides (C2–C6). Its optimal performance occurred at pH 8.5 and 50°C towards p-nitrophenyl caproate and triacetin. Sequence alignment revealed that EstOF4 shared 71% identity to esterase Est30 from Geobacillus stearothermophilus with a typical lipase pentapeptide motif G91LS93LG95. A structural model developed from homology modeling revealed that EstOF4 possessed a typical esterase 6α/7β hydrolase fold and a cap domain. Site-directed mutagenesis and inhibition studies confirmed the putative catalytic triad Ser93, Asp190 and His220.

Conclusion

EstOF4 is a new bacterial esterase with a preference to short chain ester substrates. With a high sequence identity towards esterase Est30 and several others, EstOF4 was classified into the same bacterial lipolytic family, Family XIII. All the members in this family originate from the same bacterial genus, bacillus and display optimal activities from neutral pH to alkaline conditions with short and middle chain length substrates. However, with roughly 70% sequence identity, these enzymes showed hugely different thermal stabilities, indicating their diverse thermal adaptations via just changing a few amino acid residues.     

Identification and Biochemical Studies on Novel Non-Nucleoside Inhibitors of the Enzyme Adenosine Kinase

The enzyme adenosine kinase (AK) plays a key role in the regulation of intracellular and extracellular concentration of adenosine (Ado), which exhibits potent hormonal activity in cardiovascular, nervous and immune systems. In view of the pharmacological effects of Ado, there is much interest in identifying inhibitors of AK, which can augment its tissue-protective effects. In this study, we have screened 1040 compounds from a chemical library of putative kinase inhibitors for their effect on purified human recombinant AK. These studies have identified 8 novel, non-nucleoside AK inhibitors. Four of these compounds (viz. 2-tert-butyl-4H-benzo[1,2,4]thiadiazine-3-thione (2759–0749); N-(5,6-diphenyl-furo[2,3-d]pyrimidin-4-yl)-propionamide (3998–0118); 3-[5,6-Bis-(4-methoxy-phenyl)-furo[2,3-d]pyrimidin-4-ylamino]-propan-1-ol (4072–2732); and 2-[2-(3,4-dihydroxy-phenyl)-5-phenyl-1H-imidazol-4-yl]-fluoren-9-one (8008–6198)), which inhibited human AK in a concentration-dependent manner in a low micromolar range (IC₅₀ = 0.38 ∼ 1.98 μM) were further studied. Kinetic and structural studies on these compounds provide evidence that inhibition of AK by these compounds was competitive with respect to Ado and non-competitive for ATP. All of these compounds also inhibited uptake of Ado and its metabolism in cultured mammalian cells at comparable concentrations indicating their efficient cellular penetrability. These AK inhibitors, whose chemical structures differ significantly from all previously known inhibitors, provide useful lead compounds for identification of more potent but less toxic AK inhibitors that may prove useful for therapeutic purposes.     

Further Identification of Novel Lantibiotic Operons Using LanM-Based Genome Mining

Lantibiotics are gene-encoded antimicrobial peptides that are distinguished by the presence of the unusual structures, lanthionine and β-methyllanthionine, which are introduced through enzyme-catalysed post-translational modification. Lantibiotics can be subdivided on the basis of the nature of the enzyme(s) which catalyse this reaction. Lantibiotic synthetases, generically designated LanM, which catalyse the dehydration of serines (and threonines) followed by the formation lanthionine (and β-methyllanthioine), are responsible for the synthesis of the largest subdivision, type 2. One can take advantage of the conserved nature of LanM proteins to screen for and bioinformatically characterize novel lantibiotic-encoding operons in genome-sequenced microorganisms. Having employed this strategy with success previously, here we update the investigation to reveal the existence of 124 LanM homologs encoded within genome-sequenced microbes. Further analysis focussed specifically on 9 novel lantibiotic gene clusters in Anaerocellum thermophilum DSM 6725, Anaerococcus tetradius ATCC 35098, Corynebacterium matruchotti ATCC 33806, Streptococcus suis 98HAH33, Geobacillus sp. G11MC16, Nostoc punctiforme PCC 73102 (× 2; one on plasmid and one on the chromosome) and Streptococcus pneumoniae CDC 0288-04 and TIGR4. Furthermore, screening of metagenomic datasets revealed 11 additional LanM-encoding genes from a variety of environments. The alignment of these LanM proteins facilitated a detailed investigation of conserved domains and led to the design of an improved set of degenerate primers which can be employed in the laboratory to identify strains containing type 2 lantibiotic gene clusters.     

Identification of the Dimerization Domain of Dehalogenase IVa of Burkholderia cepacia MBA4

Haloacid dehalogenases are enzymes that catalyze the hydrolytic removal of halogens from haloalkanoic acids. Dehalogenase IVa (DehIVa) from Burkholderia cepacia MBA4 and dehalogenase CI (DehCI) from Pseudomonas sp. strain CBS3 exhibit 68% identity. Despite their similarity DehIVa is a dimeric enzyme while DehCI is a monomer. In this work, we describe the identification of the domain that confers the dimerization function of DehIVa. Recombinant DNA molecules were constructed by fusion of the respective dehalogenase genes hdlIVa and dehCI. When amino acids 73 to 89 of DehCI were replaced by amino acids 74 to 90 of DehIVa, the recombinant molecule migrated like that of DehIVa in a nondenaturing activity-stained gel. Similarly, when residues 73 to 89 of DehIVa were replaced by the corresponding residues of DehCI, the chimera migrated as a monomer. These 17 amino acid changes were able to determine the aggregation states of the molecules. The retention of the catalytic function in these chimeras indicated that the overall folding of these proteins was not affected. Site-directed mutagenesis on hdlIVa however indicated that amino acids Phe58, Thr65, Leu78, and Phe92 of DehIVa are also important for the aggregation state of the protein. This indicates that the 17 residues are not sufficient for the dimerization of the protein.     

Est10: A Novel Alkaline Esterase Isolated from Bovine Rumen Belonging to the New Family XV of Lipolytic Enzymes

A metagenomic fosmid library from bovine rumen was used to identify clones with lipolytic activity. One positive clone was isolated. The gene responsible for the observed phenotype was identified by in vitro transposon mutagenesis and sequencing and was named est10. The 367 amino acids sequence harbors a signal peptide, the conserved secondary structure arrangement of alpha/beta hydrolases, and a GHSQG pentapeptide which is characteristic of esterases and lipases. Homology based 3D-modelling confirmed the conserved spatial orientation of the serine in a nucleophilic elbow. By sequence comparison, Est10 is related to hydrolases that are grouped into the non-specific Pfam family DUF3089 and to other characterized esterases that were recently classified into the new family XV of lipolytic enzymes. Est10 was heterologously expressed in Escherichia coli as a His-tagged fusion protein, purified and biochemically characterized. Est10 showed maximum activity towards C4 aliphatic chains and undetectable activity towards C10 and longer chains which prompted its classification as an esterase. However, it was able to efficiently catalyze the hydrolysis of aryl esters such as methyl phenylacetate and phenyl acetate. The optimum pH of this enzyme is 9.0, which is uncommon for esterases, and it exhibits an optimal temperature at 40°C. The activity of Est10 was inhibited by metal ions, detergents, chelating agents and additives. We have characterized an alkaline esterase produced by a still unidentified bacterium belonging to a recently proposed new family of esterases.     

Biotransformation of Explosives by the Old Yellow Enzyme Family of Flavoproteins

Several independent studies of bacterial degradation of nitrate ester explosives have demonstrated the involvement of flavin-dependent oxidoreductases related to the old yellow enzyme (OYE) of yeast. Some of these enzymes also transform the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). In this work, catalytic capabilities of five members of the OYE family were compared, with a view to correlating structure and function. The activity profiles of the five enzymes differed substantially; no one compound proved to be a good substrate for all five enzymes. TNT is reduced, albeit slowly, by all five enzymes. The nature of the transformation products differed, with three of the five enzymes yielding products indicative of reduction of the aromatic ring. Our findings suggest two distinct pathways of TNT transformation, with the initial reduction of TNT being the key point of difference between the enzymes. Characterization of an active site mutant of one of the enzymes suggests a structural basis for this difference.     

Isolation and Identification of an Endophytic Strain EJS-3 Producing Novel Fibrinolytic Enzymes

An endophytic strain EJS-3, which produces a novel fibrinolytic enzyme, was screened from root tissue of Stemona japonica (Blume) Miq, a chinese traditional medicine. This strain was identified as Paenibacillus polymyxa (DQ120522) by morphological, physiological, and biochemical tests and 16S rRNA gene sequence analysis. Two serine-type fibrinolytic enzymes with a relative molecular weight about 118 and 49 kDa, respectively, which are larger than known fibrinolytic enzymes, were found by the SDS–fibrin zymogram or by fibrin-inhibitor zymography gels. No work on P. polymyxa-producing fibrinolytic enzymes has been reported.     

Identification and Characterization of a Novel CprA Reductive Dehalogenase Specific to Highly Chlorinated Phenols from Desulfitobacterium hafniense Strain PCP-1

Desulfitobacterium hafniense strain PCP-1 reductively dechlorinates pentachlorophenol (PCP) to 3-chlorophenol and a variety of halogenated aromatic compounds at the ortho, meta, and para positions. Several reductive dehalogenases (RDases) are thought to be involved in this cascade of dehalogenation. We partially purified a novel RDase involved in the dechlorination of highly chlorinated phenols from strain PCP-1 cultivated in the presence of 2,4,6-trichlorophenol. The RDase was membrane associated, and the activity was sensitive to oxygen, with a half-life of 128 min upon exposure to air. The pH and temperature optima were 7.0 and 55°C, respectively. Several highly chlorinated phenols were dechlorinated at the ortho positions. The highest dechlorinating activity levels were observed with PCP, 2,3,4,5-tetrachlorophenol, and 2,3,4-trichlorophenol. 3-Chloro-4-hydroxyphenylacetate, 3-chloro-4-hydroxybenzoate, dichlorophenols, and monochlorophenols were not dechlorinated. The apparent K_m value for PCP was 46.7 μM at a methyl viologen concentration of 2 mM. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activity, suggesting the involvement of a corrinoid cofactor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partially purified preparation revealed 2 bands with apparent molecular masses of 42 and 47 kDa. Mass spectrometry analysis using Mascot to search the genome sequence of D. hafniense strain DCB-2 identified the 42-kDa band as NADH-quinone oxidoreductase, subunit D, and the 47-kDa band as the putative chlorophenol RDase CprA3. This is the first report of an RDase with high affinity and high dechlorinating activity toward PCP.Halogenated compounds are generally known as toxic environmental pollutants. Hydrogenolytic reductive dehalogenation, a reaction involving the replacement of one halogen atom with one hydrogen atom, is the predominant mechanism for their transformation in anaerobic environments. This process can sustain microbial growth via electron transport-coupled phosphorylation (10, 26, 31). The majority of the known reductive dehalogenases (RDases) belong to the CprA/PceA family. These are single-polypeptide membrane-associated anaerobic enzymes that are synthesized as preproteins with a cleavable twin arginine translocation (TAT) peptide signal. They contain one corrinoid and two iron-sulfur clusters as cofactors.CprA enzymes catalyzing the reductive dechlorination of chloroaromatics have been purified from Desulfitobacterium hafniense strain DCB-2 (6), Desulfitobacterium dehalogenans (30), Desulfitobacterium chlororespirans strain Co23 (12, 14), Desulfitobacterium sp. strain PCE1 (29), and D. hafniense strain PCP-1 (28) and characterized, and PceA enzymes have been purified from Sulfurospirillum multivorans (22, 23), Desulfitobacterium sp. strain PCE-S (18, 19), D. hafniense strain TCE1 (29), Dehalococcoides ethenogenes 195 (15, 16), Desulfitobacterium sp. strain PCE1 (29), Dehalobacter restrictus (17, 25), Desulfitobacterium sp. strain Y51 (27), and Dehalococcoides sp. strain VS (20) and characterized. However, none of these enzymes showed high dechlorinating activity toward highly chlorinated phenols such as pentachlorophenol (PCP).D. hafniense strain PCP-1 is the only known strict anaerobic bacterium which reductively dechlorinates PCP to 3-chlorophenol (3-CP) and a variety of halogenated aromatic compounds at the ortho, meta, and para positions (2, 7). It dechlorinates PCP at the ortho, ortho, para, and meta positions in the following order: PCP → 2,3,5,6-tetrachlorophenol (2,3,5,6-TeCP) → 3,4,5-trichlorophenol (3,4,5-TCP) → 3,5-dichlorophenol (3,5-DCP) → 3-CP (7). Several RDases are thought to operate during this sequence of dechlorinations. Two RDases have already been purified from strain PCP-1. The first one, CrdA, is a membrane-associated enzyme, not related to CprA/PceA-type RDases, that mediates ortho dechlorination of 2,4,6-TCP and several chlorophenols (3). The second enzyme, CprA5, catalyzes the meta and para dechlorination of 3,5-DCP and several chlorophenols (28). Three other putative cprA genes were identified in strain PCP-1 (cprA2, cprA3, and cprA4), which suggests that other RDases with different specificities toward halogenated compounds exist in this strain (8, 31, 32). In this study, we have partially purified and characterized a new CprA-type RDase (CprA3) from strain PCP-1. CprA3 is the first reported RDase with high affinity toward PCP and with high ortho-dechlorinating activity toward PCP and other highly chlorinated phenols.     

Identification of a Deubiquitinating Enzyme as a Novel AGS3-Interacting Protein

Activator of G protein Signaling 3 (AGS3) is a receptor-independent G protein activator that has been implicated in multiple biological events such as brain development, neuroplasticity and addiction, cardiac function, Golgi structure/function, macroautophagy and metabolism. However, how AGS3 is regulated is little known. We demonstrate here that AGS3 interacts with a ubiquitin specific protease USP9x, and this interaction is at least partially mediated through the C-terminal G protein regulatory domain of AGS3. Knockdown of USP9x causes a moderate reduction in the level of AGS3. In contrast, overexpression of either USP9x or its deubiquitinating domain UCH increases the amount of AGS3, whereas expression of the mutant UCH domain that lacks deubiquitinating activity does not have the same effect. As previously observed in AGS3 knockdown cells, the localization of several marker proteins of the late Golgi compartments is disturbed in cells depleted of USP9x. Taken together, our study suggests that USP9x can modulate the level of a subpopulation of AGS3, and this modulation plays a role in regulating the structure of the late Golgi compartments. Finally, we have found that levels of AGS3 and USP9x are co-regulated in the prefrontal cortex of rats withdrawn from repeated cocaine treatment. In conjunction with the above data, this observation indicates a potential role of USP9X in the regulation of the AGS3 level during cocaine-induced neuroplasticity.     

Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans-4-Hydroxylation of l-Pipecolic Acid

Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward l-pipecolic acid (l-Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme l-Pip trans-4-hydroxylase (Pip4H) of F. oxysporum (FoPip4H) belongs to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio- and stereoselective hydroxylation of l-Pip, and produces optically pure trans-4-hydroxy-l-pipecolic acid (trans-4-l-HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with FoPip4H, and five of these also had l-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) had a broader substrate specificity spectrum than other homologues and reacted with the l and d forms of various cyclic and aliphatic amino acids. Using FoPip4H as a biocatalyst, a system for the preparative-scale production of chiral trans-4-l-HyPip was successfully developed. Thus, we report a fungal family of l-Pip hydroxylases and the enzymatic preparation of trans-4-l-HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities.     

Analysis of the Bacteriolytic Enzymes of the Autolytic Lactococcus lactis subsp. cremoris Strain AM2 by Renaturing Polyacrylamide Gel Electrophoresis: Identification of a Prophage-Encoded Enzyme

Lactococcus lactis subsp. cremoris AM2 was previously shown to lyse early and extensively during cheese ripening (M.-P. Chapot-Chartier, C. Deniel, M. Rousseau, L. Vassal, and J.-C. Gripon, Int. Dairy J. 4:251–269, 1994). We analyzed the bacteriolytic activities of autolytic strain AM2 by using renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed with two different substrates in the gel, Micrococcus lysodeikticus and L. lactis autoclaved cells. Several lytic activities were detected in L. lactis AM2; a major lytic activity, designated A2 (46 kDa), was found only with the L. lactis cell substrate. This activity appears to be different from major peptidoglycan hydrolase AcmA characterized previously (G. Buist, J. Kok, K. J. Leenhouts, M. Dabrowska, G. Venema, and A. J. Haandrickman, J. Bacteriol. 177:1554–1563, 1995), which has a similar molecular mass. The two enzymes differ in substrate specificity as well as in sensitivity to pH and different chemical compounds. L. lactis AM2 is lysogenic and mitomycin C inducible. Enzyme A2 was shown to be inducible by mitomycin C and to be prophage encoded. It was identified as an enzyme similar to the lysin encoded by lactococcal small isometric temperate bacteriophages. A prophage-cured derivative of L. lactis AM2 was obtained, and this isolate exhibited different autolytic properties than AM2. After prolonged incubation in the stationary phase after growth on M17 medium, the extent of lysis of an AM2 culture was 60%, whereas over the same period there was almost no lysis in a prophage-cured derivative strain culture. These results suggest that the prophage lytic system is involved in the strain AM2 lysis observed in liquid medium and that it could also be involved in the lysis observed during cheese ripening.     

Evolutionary Analysis of the TPP-Dependent Enzyme Family

The evolutionary relationships of the thiamine pyrophosphate (TPP)-dependent family of enzymes was investigated by generation of a neighbor joining phylogenetic tree using sequences from the conserved pyrophosphate (PP) and pyrimidine (Pyr) binding domains of 17 TPP-dependent enzymes. This represents the most comprehensive analysis of TPP-dependent enzyme evolution to date. The phylogeny was shown to be robust by comparison with maximum likelihood trees generated for each individual enzyme and also broadly confirms the evolutionary history proposed recently from structural comparisons alone (Duggleby 2006). The phylogeny is most parsimonious with the TPP enzymes having arisen from a homotetramer which subsequently diverged into an α₂β₂ heterotetramer. The relationship between the PP- and Pyr-domains and the recruitment of additional protein domains was examined using the transketolase C-terminal (TKC)-domain as an example. This domain has been recruited by several members of the family and yet forms no part of the active site and has unknown function. Removal of the TKC-domain was found to increase activity toward β-hydroxypyruvate and glycolaldehyde. Further truncations of the Pyr-domain yielded several variants with retained activity. This suggests that the influence of TKC-domain recruitment on the evolution of the mechanism and specificity of transketolase (TK) has been minor, and that the smallest functioning unit of TK comprises the PP- and Pyr-domains, whose evolutionary histories extend to all TPP-dependent enzymes.     

Discovery of a Eukaryotic Pyrroloquinoline Quinone-Dependent Oxidoreductase Belonging to a New Auxiliary Activity Family in the Database of Carbohydrate-Active Enzymes

Pyrroloquinoline quinone (PQQ) is a redox cofactor utilized by a number of prokaryotic dehydrogenases. Not all prokaryotic organisms are capable of synthesizing PQQ, even though it plays important roles in the growth and development of many organisms, including humans. The existence of PQQ-dependent enzymes in eukaryotes has been suggested based on homology studies or the presence of PQQ-binding motifs, but there has been no evidence that such enzymes utilize PQQ as a redox cofactor. However, during our studies of hemoproteins, we fortuitously discovered a novel PQQ-dependent sugar oxidoreductase in a mushroom, the basidiomycete Coprinopsis cinerea. The enzyme protein has a signal peptide for extracellular secretion and a domain for adsorption on cellulose, in addition to the PQQ-dependent sugar dehydrogenase and cytochrome domains. Although this enzyme shows low amino acid sequence homology with known PQQ-dependent enzymes, it strongly binds PQQ and shows PQQ-dependent activity. BLAST search uncovered the existence of many genes encoding homologous proteins in bacteria, archaea, amoebozoa, and fungi, and phylogenetic analysis suggested that these quinoproteins may be members of a new family that is widely distributed not only in prokaryotes, but also in eukaryotes.     

Strategy for Stabilizing Enzymes Part Two: Increasing Enzyme Stability by Selective Chemical Modification

The molecular mechanisms of change in the thermal stability of proteins modified with low molecular weight reagents are discussed. The choice of stabilization mechanisms to be used as a general strategy for increasing enzyme stability by chemical modification is addressed. Hydrophilization of nonpolar surface areas is the most simple and reliable approach to artificial stabilization of enzymes for use in applied biochemistry and biotechnology.     

dEMBF: A Comprehensive Database of Enzymes of Microalgal Biofuel Feedstock

Microalgae have attracted wide attention as one of the most versatile renewable feedstocks for production of biofuel. To develop genetically engineered high lipid yielding algal strains, a thorough understanding of the lipid biosynthetic pathway and the underpinning enzymes is essential. In this work, we have systematically mined the genomes of fifteen diverse algal species belonging to Chlorophyta, Heterokontophyta, Rhodophyta, and Haptophyta, to identify and annotate the putative enzymes of lipid metabolic pathway. Consequently, we have also developed a database, dEMBF (Database of Enzymes of Microalgal Biofuel Feedstock), which catalogues the complete list of identified enzymes along with their computed annotation details including length, hydrophobicity, amino acid composition, subcellular location, gene ontology, KEGG pathway, orthologous group, Pfam domain, intron-exon organization, transmembrane topology, and secondary/tertiary structural data. Furthermore, to facilitate functional and evolutionary study of these enzymes, a collection of built-in applications for BLAST search, motif identification, sequence and phylogenetic analysis have been seamlessly integrated into the database. dEMBF is the first database that brings together all enzymes responsible for lipid synthesis from available algal genomes, and provides an integrative platform for enzyme inquiry and analysis. This database will be extremely useful for algal biofuel research. It can be accessed at http://bbprof.immt.res.in/embf.