Novel transmembrane lipases of alpha/beta hydrolase fold

Processing of exogenous glycerol esters is an initial step in energy derivation for many bacterial cells. Lipid-rich environments settled by a variety of organisms exert strong evolutionary pressure for establishing enzymatic pathways involved in lipid metabolism. However, a certain number of enzymes involved in this process remain unknown since they do not share detectable sequence similarity with any known protein domains. Using distant homology detection and fold recognition we predict that bacterial transmembrane proteins belonging to the uncharacterized domain of unknown function 2319 (DUF2319) family possess the alpha/beta hydrolase fold domain together with the catalytic triad critical for hydrolysis. A detailed analysis of sequence/structure features and genomic context indicates that DUF2319 proteins may be involved in lipid metabolism. Therefore, these enzymes are likely to serve as extracellular lipases.     

Murine UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase lacking the gamma-subunit retains substantial activity toward acid hydrolases

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on acid hydrolases. The transferase exists as an alpha(2)beta(2)gamma(2) hexameric complex with the alpha- and beta-subunits derived from a single precursor molecule. The catalytic function of the transferase is attributed to the alpha- and beta-subunits, whereas the gamma-subunit is believed to be involved in the recognition of a conformation-dependent protein determinant common to acid hydrolases. Using knock-out mice with mutations in either the alpha/beta gene or the gamma gene, we show that disruption of the alpha/beta gene completely abolishes phosphorylation of high mannose oligosaccharides on acid hydrolases whereas knock-out of the gamma gene results in only a partial loss of phosphorylation. These findings demonstrate that the alpha/beta-subunits, in addition to their catalytic function, have some ability to recognize acid hydrolases as specific substrates. This process is enhanced by the gamma-subunit.     

Identification of a novel class in the alpha/beta hydrolase fold superfamily: the N-myc differentiation-related proteins

The alpha/beta hydrolases constitute a large protein superfamily that mainly consists of enzymes that catalyze a diverse range of reactions. These proteins exhibit the alpha/beta hydrolase fold, the essential features of which have recently been delineated: the presence of at least five parallel beta-strands, a catalytic triad in a specific order (nucleophile-acid-histidine), and a nucleophilic elbow. Because of the difficulties experimentally in identifying protein structures, we have used a Bayesian computational algorithm (PROBE) to identify the members of this superfamily based on distant sequence relationships. We found that the presence of five sequence motifs, which contain residues important for substrate binding and stabilization of the fold, are required for membership in this superfamily. The superfamily consists of at least 909 members, including the N-myc downstream regulated proteins, which are believed to be involved in cell differentiation. Unlike most of the other superfamily members, the N-myc downstream regulated proteins have never been proposed to possess the alpha/beta hydrolase fold and do not appear to be hydrolases.     

Bacterial 2,4-Dioxygenases: New Members of the α/β Hydrolase-Fold Superfamily of Enzymes Functionally Related to Serine Hydrolases

1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) from Pseudomonas putida 33/1 and 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) from Arthrobacter ilicis Rü61a catalyze an N-heterocyclic-ring cleavage reaction, generating N-formylanthranilate and N-acetylanthranilate, respectively, and carbon monoxide. Amino acid sequence comparisons between Qdo, Hod, and a number of proteins belonging to the alpha/beta hydrolase-fold superfamily of enzymes and analysis of the similarity between the predicted secondary structures of the 2,4-dioxygenases and the known secondary structure of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 strongly suggested that Qdo and Hod are structurally related to the alpha/beta hydrolase-fold enzymes. The residues S95 and H244 of Qdo were found to be arranged like the catalytic nucleophilic residue and the catalytic histidine, respectively, of the alpha/beta hydrolase-fold enzymes. Investigation of the potential functional significance of these and other residues of Qdo through site-directed mutagenesis supported the hypothesis that Qdo is structurally as well as functionally related to serine hydrolases, with S95 being a possible catalytic nucleophile and H244 being a possible catalytic base. A hypothetical reaction mechanism for Qdo-catalyzed 2,4-dioxygenolysis, involving formation of an ester bond between the catalytic serine residue and the carbonyl carbon of the substrate and subsequent dioxygenolysis of the covalently bound anionic intermediate, is discussed.     

NMR structure of the conserved hypothetical protein TM0487 from Thermotoga maritima: implications for 216 homologous DUF59 proteins

The NMR structure of the conserved hypothetical protein TM0487 from Thermotoga maritima represents an alpha/beta-topology formed by the regular secondary structures alpha1-beta1-beta2-alpha2-beta3-beta4-alpha3- beta5-3(10)-alpha4, with a small anti-parallel beta-sheet of beta-strands 1 and 2, and a mixed parallel/anti-parallel beta-sheet of beta-strands 3-5. Similar folds have previously been observed in other proteins, with amino acid sequence identity as low as 3% and a variety of different functions. There are also 216 sequence homologs of TM0487, which all have the signature sequence of domains of unknown function 59 (DUF59), for which no three-dimensional structures have as yet been reported. The TM0487 structure thus presents a platform for homology modeling of this large group of DUF59 proteins. Conserved among most of the DUF59s are 13 hydrophobic residues, which are clustered in the core of TM0487. A putative active site of TM0487 consisting of residues D20, E22, L23, T51, T52, and C55 is conserved in 98 of the 216 DUF59 sequences. Asp20 is buried within the proposed active site without any compensating positive charge, which suggests that its pK(a) value may be perturbed. Furthermore, the DUF59 family includes ORFs that are part of a conserved chromosomal group of proteins predicted to be involved in Fe-S cluster metabolism.     

Furanosidase superfamily: search of homologues

The furanosidase superfamily contains GH32, GH43, GH62, GH68, GH117, DUF377, and DUF1861 families of glycoside hydrolases and their homologues. Catalytic domains of these families have five-bladed beta-propeller tertiary structure. Iterative screening of the protein database allowed to support their relationship as well as evolutionary connections with domains from GH33 and GH93 families of glycoside hydrolases. The latter two have structure of the six-bladed beta-propeller. Among revealed homologues we found 441 unclassified proteins. These proteins are combined into 39 groups based on homology: FURAN1-FURAN39. FURAN8 and FURAN36 can be considered as separate subfamilies within GH43 and GH32 families of glycoside hydrolases, respectively. The remaining 37 groups are new families of hypothetical glycoside hydrolases.     

Furanosidase superfamily: Search of homologues

The furanosidase superfamily contains the GH32, GH43, GH62, GH68, GH117, DUF377 (GH130), and DUF1861 families of glycoside hydrolases and their homologues. Catalytic domains of these families have five-bladed β-propeller tertiary structure. Iterative screening of the protein database supports of their relationship as well as evolutionary connections with domains from GH33 and GH93 families of glycoside hydrolases. The latter two have the structure of the six-bladed β-propeller. Among detected homologues we found 441 unclassified proteins. These proteins are combined into 39 groups based on homology: FURAN1-FURAN39. FURAN8 and FURAN36 can be considered as separate subfamilies within the GH43 and GH32 families of glycoside hydrolases, respectively. The remaining 37 groups are new families of hypothetical glycoside hydrolases.     

aCHEdb: the database system for ESTHER, the alpha/beta fold family of proteins and the Cholinesterase gene server.

Acetylcholinesterase belongs to a family of proteins, the alpha/beta hydrolase fold family, whose constituents evolutionarily diverged from a common ancestor and share a similar structure of a central beta sheet surrounded by alpha helices. These proteins fulfil a wide range of physiological functions (hydrolases, adhesion molecules, hormone precursors) [Krejci,E., Duval,N., Chatonnet,A., Vincens,P. and Massoulié,J. (1991) Proc. Natl. Acad. Sci. USA , 88, 6647-6651]. ESTHER (for esterases, alpha/beta hydrolase enzymes and relatives) is a database aimed at collecting in one information system, sequence data together with biological annotations and experimental biochemical results related to the structure-function analysis of the enzymes of the family. The major upgrade of the database comes from the use of a new database management system: aCHEdb which uses the ACeDB program designed by Richard Durbin and Jean Thierry-Mieg. It can be found at http://www.ensam.inra.fr/cholinesterase     

Cloning and tissue distribution of three murine alpha/beta hydrolase fold protein cDNAs

We have cloned 3 novel murine cDNAs encoding proteins containing an alpha/beta hydrolase fold; a catalytic domain found in a very wide range of enzymes. These proteins belong to the prosite UPF0017 uncharacterized protein family and we have named them lung alpha/beta hydrolase 1, 2, and 3 (LABH) since they were cloned from lung cDNA. All have 9 coding exons, encoding 412, 425, and 411 residue proteins respectively (46-48 kDa); LABH1 being closely related to LABH3 having 45% identity. All 3 proteins have a single predicted amino-terminus transmembrane domain. An alignment of family members from different phyla enabled the identification of the LABH1 catalytic triad as Ser211, Asp337, and His366. mRNA expression levels of LABH1 and 3 were highest in liver and LABH2 highest in testis. These findings suggest that the LABH proteins consist of a novel family of membrane bound enzymes whose function has yet to be determined.     

The structure of Mycobacterium tuberculosis MPT51 (FbpC1) defines a new family of non-catalytic alpha/beta hydrolases

Mycobacterium tuberculosis, the causative agent of tuberculosis, is known to secrete a number of highly immunogenic proteins that are thought to confer pathogenicity, in part, by mediating binding to host tissues. Among these secreted proteins are the trimeric antigen 85 (Ag85) complex and the related MPT51 protein, also known as FbpC1. While the physiological function of Ag85, a mycolyltransferase required for the biosynthesis of the cell wall component alpha,alpha'-trehalose dimycolate (or cord factor), has been identified recently, the function of the closely related MPT51 (approximately 40% identity with the Ag85 components) remains to be established. The crystal structure of M.tuberculosis MPT51, determined to 1.7 A resolution, shows that MPT51, like the Ag85 components Ag85B and Ag85C2, folds as an alpha/beta hydrolase, but it does not contain any of the catalytic elements required for mycolyltransferase activity. Moreover, the absence of a recognizable alpha,alpha'-trehalose monomycolate-binding site and the failure to detect an active site suggest that the function of MPT51 is of a non-enzymatic nature and that MPT51 may in fact represent a new family of non-catalytic alpha/beta hydrolases. Previous experimental evidence and the structural similarity to some integrins and carbohydrate-binding proteins led to the hypothesis that MPT51 might have a role in host tissue attachment, whereby ligands may include the serum protein fibronectin and small sugars.     

Relation between domain evolution, specificity, and taxonomy of the alpha-amylase family members containing a C-terminal starch-binding domain.

The alpha-amylase family (glycoside hydrolase family 13; GH 13) contains enzymes with approximately 30 specificities. Six types of enzyme from the family can possess a C-terminal starch-binding domain (SBD): alpha-amylase, maltotetraohydrolase, maltopentaohydrolase, maltogenic alpha-amylase, acarviose transferase, and cyclodextrin glucanotransferase (CGTase). Such enzymes are multidomain proteins and those that contain an SBD consist of four or five domains, the former enzymes being mainly hydrolases and the latter mainly transglycosidases. The individual domains are labelled A [the catalytic (beta/alpha)8-barrel], B, C, D and E (SBD), but D is lacking from the four-domain enzymes. Evolutionary trees were constructed for domains A, B, C and E and compared with the 'complete-sequence tree'. The trees for domains A and B and the complete-sequence tree were very similar and contain two main groups of enzymes, an amylase group and a CGTase group. The tree for domain C changed substantially, the separation between the amylase and CGTase groups being shortened, and a new border line being suggested to include the Klebsiella and Nostoc CGTases (both four-domain proteins) with the four-domain amylases. In the 'SBD tree' the border between hydrolases (mainly alpha-amylases) and transglycosidases (principally CGTases) was not readily defined, because maltogenic alpha-amylase, acarviose transferase, and the archaeal CGTase clustered together at a distance from the main CGTase cluster. Moreover the four-domain CGTases were rooted in the amylase group, reflecting sequence relationships for the SBD. It appears that with respect to the SBD, evolution in GH 13 shows a transition in the segment of the proteins C-terminal to the catalytic (beta/alpha)8-barrel(domain A).     

Characterization of a novel β-L-Arabinofuranosidase in Bifidobacterium longum: functional elucidation of A DUF1680 family member

Pfam DUF1680 (PF07944) is an uncharacterized protein family conserved in many species of bacteria, actinomycetes, fungi, and plants. In a previous article, we cloned and characterized the hypBA2 gene as a β-l-arabinobiosidase in Bifidobacterium longum JCM 1217. In this study, we cloned a DUF1680 family member, the hypBA1 gene, which constitutes a gene cluster with hypBA2. HypBA1 is a novel β-l-arabinofuranosidase that liberates l-arabinose from the l-arabinofuranose (Araf)-β1,2-Araf disaccharide. HypBA1 also transglycosylates 1-alkanols with retention of the anomeric configuration. Mutagenesis and azide rescue experiments indicated that Glu-366 is a critical residue for catalytic activity. This report provides the first characterization of a DUF1680 family member, which defines a new family of glycoside hydrolases, the GH family 127.     

Structural comparisons of TIM barrel proteins suggest functional and evolutionary relationships between beta-galactosidase and other glycohydrolases

Beta-galactosidase (lacZ) from Escherichia coli is a 464 kDa homotetramer. Each subunit consists of five domains, the third being an alpha/beta barrel that contains most of the active site residues. A comparison is made between each of the domains and a large set of proteins representative of all structures from the protein data bank. Many structures include an alpha/beta barrel. Those that are most similar to the alpha/beta barrel of E. coli beta-galactosidase have similar catalytic residues and belong to the so-called "4/7 superfamily" of glycosyl hydrolases. The structure comparison suggests that beta-amylase should also be included in this family. Of three structure comparison methods tested, the "ProSup" procedure of Zu-Kang and Sippl and the "Superimpose" procedure of Diederichs were slightly superior in discriminating the members of this superfamily, although all procedures were very powerful in identifying related protein structures. Domains 1, 2, and 4 of E. coli beta-galactosidase have topologies related to "jelly-roll barrels" and "immunoglobulin constant" domains. This fold also occurs in the cellulose binding domains (CBDs) of a number of glycosyl hydrolases. The fold of domain 1 of E. coli beta-galactosidase is closely related to some CBDs, and the domain contributes to substrate binding, but in a manner unrelated to cellulose binding by the CBDs. This is typical of domains 1, 2, 4, and 5, which appear to have been recruited to play roles in beta-galactosidase that are unrelated to the functions that such domains provide in other contexts. It is proposed that beta-galactosidase arose from a prototypical single domain alpha/beta barrel with an extended active site cleft. The subsequent incorporation of elements from other domains could then have reduced the size of the active site from a cleft to a pocket to better hydrolyze the disaccharide lactose and, at the same time, to facilitate the production of inducer, allolactose.     

Serine carboxypeptidase-like acyltransferases

In plant secondary metabolism, an alternative pathway of ester formation is facilitated by acyltransferases accepting 1-O-beta-acetal esters (1-O-beta-glucose esters) as acyl donors instead of coenzyme A thioesters. Molecular data indicate homology of these transferases with hydrolases of the serine carboxypeptidase type defining them as serine carboxypeptidase-like (SCPL) acyltransferases. During evolution, they apparently have been recruited from serine carboxypeptidases and adapted to take over acyl transfer function. SCPL acyltransferases belong to the highly divergent class of alpha/beta hydrolases. These enzymes make use of a catalytic triad formed by a nucleophile, an acid and histidine acting as a charge relay system for the nucleophilic attack on amide or ester bonds. In analogy to SCPL acyltransferases, bacterial thioesterase domains are known which favour transferase activity over hydrolysis. Structure elucidation reveals water exclusion and a distortion of the oxyanion hole responsible for the changed activity. In plants, SCPL proteins form a large family. By sequence comparison, a distinguished number of Arabidopsis SCPL proteins cluster with proven SCPL acyltransferases. This indicates the occurrence of a large number of SCPL proteins co-opted to catalyse acyltransfer reactions. SCPL acyltransferases are ideal systems to investigate principles of functional adaptation and molecular evolution of plant genes.     

Purification and characterization of casein kinase II (CKII) from delta cka1 delta cka2 Saccharomyces cerevisiae rescued by Drosophila CKII subunits. The free catalytic subunit of casein kinase II is not toxic in vivo.

Casein kinase II (CKII) is composed of a catalytic (alpha) and a regulatory (beta) subunit which unite to form an alpha 2 beta 2 holoenzyme. Saccharomyces cerevisiae CKII consists of two distinct catalytic (Sc alpha and Sc alpha') and regulatory (Sc beta and Sc beta') subunits. Simultaneous disruption of the CKA1 and CKA2 genes (encoding the alpha and alpha' subunits, respectively) is lethal. Such double disruptions can be rescued by GAL1, 10-induced expression of the Drosophila alpha and beta subunits (Dm alpha+beta) together or by GAL10-induced expression of the Drosophila alpha subunit (Dm alpha) alone (Padmanabha, R., Chen-Wu, J. L.-P., Hanna, D. E., and Glover, C. V. C. (1990) Mol. Cell. Biol. 10, 4089-4099). Here we report quantitation, purification, and characterization of casein kinase II activity from such rescued strains. Casein kinase II activity from a strain rescued by Dm alpha alone purifies as a free, catalytically active alpha subunit monomer, whereas that from a strain rescued by Dm alpha/beta purifies as a mixture of tetrameric holoenzyme and monomeric alpha subunit. Interestingly, neither Sc beta nor Sc beta' is present at detectable levels in the enzyme obtained from either strain, raising the possibility that rescue by Dm alpha alone may be mediated via the free, monomeric catalytic subunit. Overexpression of total casein kinase II activity from 6- to 18-fold is not toxic and indeed has no overt phenotypic consequences. Production of large amounts of free catalytic subunit also appears to be without effect, even though free catalytic subunit is normally undetectable in S. cerevisiae.     

Phosphorylation of casein kinase II

Casein kinase II from rabbit reticulocytes is a tetramer with an alpha,alpha' beta 2 or alpha 2 beta 2 structure; the alpha subunits contain the catalytic activity, and the beta subunits are regulatory in nature [Traugh, J.A., Lin, W. J., Takada-Axelrod, F., & Tuazon, P. T. (1990) Adv. Second Messenger Phosphoprotein Res. 24, 224-229]. When casein kinase II is isolated from rabbit reticulocytes by a rapid two-step purification of the enzyme, both the alpha and beta subunits are phosphorylated to a significant extent. In vitro, purified casein kinase II undergoes autophosphorylation on the beta subunit. In the presence of polylysine and polyarginine, phosphorylation of the beta subunits is inhibited, and the alpha subunits (alpha and alpha') become autophosphorylated. The effectiveness of polylysine coincides with the molecular weight. With basic proteins, including a number of histones and protamine, autophosphorylation of both subunits is observed. With histones, autophosphorylation of each subunit can be greater than that observed with the autophosphorylated enzyme alone or with a basic polypeptide. Thus, the potential exists for modulatory proteins to alter the autophosphorylation state of casein kinase II. Taken together, the data suggest that phosphorylation of the alpha subunit of casein kinase II in vivo may be due to an unidentified protein kinase or due to autophosphorylation. In the latter instance, casein kinase II could be transiently associated with specific intracellular compounds, such as basic proteins, with a resultant stimulation of autophosphorylation.     

More compact protein globules exhibit slower folding rates

We have demonstrated that, among proteins of the same size, alpha/beta proteins have on the average a greater number of contacts per residue due to their more compact (more "spherical") structure, rather than due to tighter packing. We have examined the relationship between the average number of contacts per residue and folding rates in globular proteins according to general protein structural class (all-alpha, all-beta, alpha/beta, alpha+beta). Our analysis demonstrates that alpha/beta proteins have both the greatest number of contacts and the slowest folding rates in comparison to proteins from the other structural classes. Because alpha/beta proteins are also known to be the oldest proteins, it can be suggested that proteins have evolved to pack more quickly and into looser structures.     

Determinants of the nucleolar targeting of protein phosphatase-1

The ubiquitously expressed protein Ser/Thr phosphatase-1 isoforms PP1alpha, PP1beta and PP1gamma1 are dynamically targeted to distinct, but overlapping cellular compartments by associated proteins. Within the nucleus of HeLa cells, EGFP-tagged PP1gamma1 and PP1beta were predominantly targeted to the nucleoli, while PP1alpha showed a more diffuse distribution. Using PP1 chimaeras and point mutants we show here that a single N-terminal residue, i.e., Gln20 for PP1alpha, Arg19 for PP1beta and Arg20 for PP1gamma1 accounts for their distinct subnuclear distribution. Our data also suggest that the N-terminus of PP1beta and PP1gamma1 harbours an interaction site for one or more nucleolar interactors.