Evolution of functional diversity in the cupin superfamily

The cupin superfamily of proteins is among the most functionally diverse of any described to date. It was named on the basis of the conserved β-barrel fold (‘cupa’ is the Latin term for a small barrel), and comprises both enzymatic and non-enzymatic members, which have either one or two cupin domains. Within the conserved tertiary structure, the variety of biochemical function is provided by minor variation of the residues in the active site and the identity of the bound metal ion. This review discusses the advantages of this particular scaffold and provides an evolutionary analysis of 18 different subclasses within the cupin superfamily.     

Potential and capabilities of hydroxynitrile lyases as biocatalysts in the chemical industry

The application of hydroxynitrile lyases (HNLs) as catalysts for the stereoselective condensation of HCN with carbonyl compounds has been reported as early as 1908. This enzymatic C-C bond coupling reaction furnishes enantiopure cyanohydrins which serve as versatile bifunctional building blocks for chemical synthesis. Screening of natural sources led to the discovery of both (R)- and (S)-selective HNLs, and several distinctly different classes of these enzymes with substantial differences concerning sequence, structure, and mechanism have been found. Especially during the last two centuries, HNLs have been developed into valuable biocatalysts, which can be produced in recombinant form by overexpression in microbial hosts, resulting in the implementation of industrial processes utilizing these enzymes. Recently, protein engineering in combination with in silico methods gave rise to the development of a tailor-made HNL for large-scale manufacturing of a specific target cyanohydrin.     

Characterization of proteins belonging to the CHAP-related superfamily within the Firmicutes

Cell division is a dynamic process ending by separation of the daughter cells. This final step requires the cleavage of the murein septum synthetized during cell division. In Streptococcus thermophilus, cse plays an important role in cell separation. Cse protein contains, at its N-terminal end, a signal peptide and a putative LysM motif suggesting that it is secreted and able to bind to the cell wall. Furthermore, the C-terminus of Cse carries a putative cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain conferring to the protein a potential catalytic activity. To gain insight into the role of Cse in the cell division process, in silico analysis of the Firmicutes proteins displaying CHAP-related domain was undertaken. This work allowed us to distinguish and characterize within the Firmicutes the 2 families of proteins (CHAP and NlpC/p60) belonging to the CHAP superfamily. These 2 families regroup mainly peptidoglycan hydrolases. Data from the literature indicate that NlpC/p60 and CHAP proteins cleave distinct peptidoglycan bonds. Among the enzymes characterized within the Firmicutes, NlpC/p60 proteins are gamma-D-glutamate-meso-diaminopimelate muropeptidase. Instead, CHAP enzymes involved in cell separation are N-acetylmuramoyl-L-alanine amidase and CHAP lysins have endopeptidase activity.     

A new dimethylsulfoniopropionate lyase of the cupin superfamily in marine bacteria

Dimethylsulfoniopropionate (DMSP) is a marine organosulfur compound with important roles in stress protection, marine biogeochemical cycling, chemical signalling and atmospheric chemistry. Diverse marine microorganisms catabolize DMSP via DMSP lyases to generate the climate-cooling gas and info-chemical dimethyl sulphide. Abundant marine heterotrophs of the Roseobacter group (MRG) are well known for their ability to catabolize DMSP via diverse DMSP lyases. Here, a new DMSP lyase DddU within the MRG strain Amylibacter cionae H-12 and other related bacteria was identified. DddU is a cupin superfamily DMSP lyase like DddL, DddQ, DddW, DddK and DddY, but shares <15% amino acid sequence identity with these enzymes. Moreover, DddU proteins forms a distinct clade from these other cupin-containing DMSP lyases. Structural prediction and mutational analyses suggested that a conserved tyrosine residue is the key catalytic amino acid residue in DddU. Bioinformatic analysis indicated that the dddU gene, mainly from Alphaproteobacteria, is widely distributed in the Atlantic, Pacific, Indian and polar oceans. For reference, dddU is less abundant than dddP, dddQ and dddK, but much more frequent than dddW, dddY and dddL in marine environments. This study broadens our knowledge on the diversity of DMSP lyases, and enhances our understanding of marine DMSP biotransformation.     

Reaction mechanism of hydroxynitrile lyases of the alpha/beta-hydrolase superfamily: the three-dimensional structure of the transient enzyme-substrate complex certifies the crucial role of LYS236

The hydroxynitrile lyases (HNLs) from Hevea brasiliensis (HbHNL) and from Manihot esculenta (MeHNL) are both members of the alpha/beta-hydrolase superfamily. Mechanistic proposals have been put forward in the past for both enzymes; they differed with respect to the role of the active-site lysine residue for which a catalytic function was claimed for the Hevea enzyme but denied for the Manihot enzyme. We applied a freeze-quench method to prepare crystals of the complex of HbHNL with the biological substrate acetone cyanohydrin and determined its three-dimensional structure. Site-directed mutagenesis was used to prepare the mutant K236L, which is inactive although its three-dimensional structure is similar to the wild-type enzyme. However, the structure of the K236L-acetone cyanohydrin complex shows the substrate in a different orientation from the wild-type complex. Finite difference Poisson-Boltzmann calculations show that in the absence of Lys(236) the catalytic base His(235) would be protonated at neutral pH. All of this suggests that Lys(236) is instrumental for catalysis in several ways, i.e. by correctly positioning the substrate, by stabilizing the negatively charged reaction product CN(-), and by modulating the basicity of the catalytic base. These data complete the elucidation of the reaction mechanism of alpha/beta-hydrolase HNLs, in which the catalytic triad acts as a general base rather than as a nucleophile; proton abstraction from the substrate is performed by the serine, and reprotonation of the product cyanide is performed by the histidine residues. Together with a threonine side chain, the active-site serine and lysine are also involved in substrate binding.     

Identification of two novel RanGTP-binding proteins belonging to the importin beta superfamily

Nucleo-cytoplasmic transport comprises a large number of distinct pathways, many of which are defined by members of the importin beta superfamily of nuclear transport receptors. These transport receptors all directly interact with RanGTP to modulate the compartment-specific binding of their transport substrates. To identify new members of the importin beta family, we used affinity chromatography on immobilized RanGTP and isolated Ran-binding protein (RanBP) 16 from HeLa cell extracts. RanBP16 and its close human homologue, RanBP17, are distant members of the importin beta family. Like the other members of the transport receptor superfamily, RanBP16 interacts with the nuclear pore complex and is able to enter the nucleus independent of energy and additional nuclear transport receptors.     

Thiol dioxygenases: unique families of cupin proteins

Proteins in the cupin superfamily have a wide range of biological functions in archaea, bacteria and eukaryotes. Although proteins in the cupin superfamily show very low overall sequence similarity, they all contain two short but partially conserved cupin sequence motifs separated by a less conserved intermotif region that varies both in length and amino acid sequence. Furthermore, these proteins all share a common architecture described as a six-stranded β-barrel core, and this canonical cupin or “jelly roll” β-barrel is formed with cupin motif 1, the intermotif region, and cupin motif 2 each forming two of the core six β-strands in the folded protein structure. The recently obtained crystal structures of cysteine dioxygenase (CDO), with contains conserved cupin motifs, show that it has the predicted canonical cupin β-barrel fold. Although there had been no reports of CDO activity in prokaryotes, we identified a number of bacterial cupin proteins of unknown function that share low similarity with mammalian CDO and that conserve many residues in the active-site pocket of CDO. Putative bacterial CDOs predicted to have CDO activity were shown to have similar substrate specificity and kinetic parameters as eukaryotic CDOs. Information gleaned from crystal structures of mammalian CDO along with sequence information for homologs shown to have CDO activity facilitated the identification of a CDO family fingerprint motif. One key feature of the CDO fingerprint motif is that the canonical metal-binding glutamate residue in cupin motif 1 is replaced by a cysteine (in mammalian CDOs) or by a glycine (bacterial CDOs). The recent report that some putative bacterial CDO homologs are actually 3-mercaptopropionate dioxygenases suggests that the CDO family may include proteins with specificities for other thiol substrates. A paralog of CDO in mammals was also identified and shown to be the other mammalian thiol dioxygenase, cysteamine dioxygenase (ADO). A tentative fingerprint motif for ADOs, or DUF1637 family members, is proposed. In ADOs, the conserved glutamate residue in cupin motif 1 is replaced by either glycine or valine. Both ADOs and CDOs appear to represent unique clades within the cupin superfamily.     

Two types of bacterial alginate lyases.

The extracellular alginate lyases were purified from Vibrio harveyi AL-128 and V. alginolyticus ATCC 17749. The former enzyme appears to be specific for alpha-1,4 bonds involving L-guluronate units in alginate, whereas the latter exhibits specificity for beta-1,4 bonds involving D-mannuronate units. The molecular weights of the enzymes were estimated to be 57,000 and 47,000, and they had isoelectric points of 4.3 and 4.6, respectively. The enzyme from strain AL-128 was most active at NaCl concentrations of 0.3 to 1.0 M. Optimum activity of the enzyme from strain ATCC 17749 was found in the presence of 5 to 10 mM CaCl2.     

Two types of bacterial alginate lyases.

The extracellular alginate lyases were purified from Vibrio harveyi AL-128 and V. alginolyticus ATCC 17749. The former enzyme appears to be specific for alpha-1,4 bonds involving L-guluronate units in alginate, whereas the latter exhibits specificity for beta-1,4 bonds involving D-mannuronate units. The molecular weights of the enzymes were estimated to be 57,000 and 47,000, and they had isoelectric points of 4.3 and 4.6, respectively. The enzyme from strain AL-128 was most active at NaCl concentrations of 0.3 to 1.0 M. Optimum activity of the enzyme from strain ATCC 17749 was found in the presence of 5 to 10 mM CaCl2.     

Molecular and genetic characterization of two pollen-expressed genes that have sequence similarity to pectate lyases of the plant pathogen Erwinia

A set of cDNAs that are expressed in tomato anthers were isolated [24]. We further characterized two of these cDNAs (LAT56 and LAT59) and their corresponding genomic clones. LAT56 and LAT59 show low levels of steady-state mRNA in immature anthers and maximal levels in mature anthers and pollen. The LAT56 and LAT59 genes are single-copy in the tomato genome, and are linked on chromosome 3, approximately 5 cM apart. Although these cDNAs did not cross-hybridize, their deduced protein sequences (P56 and P59) have 54% amino acid identity. The LAT56 and LAT59 genes each have two introns, but they are located in non-homologous positions. P56 and P59 show significant protein sequence similarity to pectate lyases of plant pathogenic bacteria. The similarity of P56 and P59 to the bacterial pectate lyases is equivalent to the homology described for different pectate lyase sequences of the genus Erwinia. We suggest that the pollen expression of LAT56 and LAT59 might relate to a requirement for pectin degradation during pollen tube growth.Abbreviations LAT, late anther tomato - bp, base pairs - MA, mature anther - PL, pectate lyase - kb, kilobase (pairs)     

A superfamily of archaeal, bacterial, and eukaryotic proteins homologous to animal transglutaminases.

Computer analysis using profiles generated by the PSI-BLAST program identified a superfamily of proteins homologous to eukaryotic transglutaminases. The members of the new protein superfamily are found in all archaea, show a sporadic distribution among bacteria, and were detected also in eukaryotes, such as two yeast species and the nematode Caenorhabditis elegans. Sequence conservation in this superfamily primarily involves three motifs that center around conserved cysteine, histidine, and aspartate residues that form the catalytic triad in the structurally characterized transglutaminase, the human blood clotting factor XIIIa'. On the basis of the experimentally demonstrated activity of the Methanobacterium phage pseudomurein endoisopeptidase, it is proposed that many, if not all, microbial homologs of the transglutaminases are proteases and that the eukaryotic transglutaminases have evolved from an ancestral protease.     

Characterization and cloning of a Tenebrio molitor hemolymph protein with sequence similarity to insect odorant-binding proteins

The yellow mealworm beetle, Tenebrio molitor, produces a number of moderately abundant low molecular weight hemolymph proteins ( approximately 12 kDa) which behave in a similar manner during purification and share antigenic epitopes. The cDNA sequence of the major component (THP12) was determined and the deduced protein sequence was found to be similar to those of insect odorant-binding proteins. Southern blot analysis suggests that at least some of the diversity in this family of proteins is encoded at the gene level. Both northern and western blot analysis indicate that THP12 is present in a variety of developmental stages and both sexes. THP12 was originally classified as an antifreeze protein, but the lack of antifreeze activity in the recombinant protein, as well as the clear separation of the antifreeze activity from THP12 following HPLC purification, has ruled out this function. The abundance of THP12, the similarity of THP12 to insect odorant-binding proteins, and the presence of hydrophobic cavities inside the protein (Rothemund et al., A new class of hexahelical insect proteins revealed as putative carriers of small hydrophobic ligands. Structure, 7 (1999) 1325-1332.) suggest that THP12 may function to carry non-water soluble compounds in the hemolymph. THP12 is also similar, particularly in structurally important regions, to other insect proteins from non-sensory tissues, suggesting the existence of a large family of carrier proteins which may perform diverse functions throughout the insect.     

Characterization of two high molecular weight proteins immunoprecipitated with an antibody against rat liver cytochrome c oxidase

Isolated rat hepatocytes were labelled with [35S]methionine, dissolved in Triton X-100-containing buffer, and incubated with antibodies against rat liver cytochrome c oxidase. After separation by dodecyl sulfate-gel electrophoresis the fluorogram of immunoprecipitated proteins showed two labelled bands with apparent molecular weights of 52000 and 182000. The immunological relationship of the two proteins to cytochrome c oxidase was demonstrated by immunocompetition with the isolated enzyme and with purified subunits IV-VIII. Although the precursor nature of the two described proteins for cytoplasmically synthesized subunits of cytochrome c oxidase cannot be excluded, the following observations do not support this assumption: 1) The amount of incorporated radioactivity is too high; 2) they are exclusively located with the microsomal fraction; 3) the turnover is rather slow, compared to that of known precursor proteins.     

Streptomyces antibioticus contains at least three oleandomycin-resistance determinants, one of which shows similarity with proteins of the ABC-transporter superfamily

Three different DNA fragments of an oleandomycin producer, Streptomyces antibioticus, conferring oleandomycin resistance were cloned in plasmid pIJ702 and expressed in Streptomyces lividans and in Streptomyces albus. These oleandomycin resistance determinants were designated as oleA (pOR400), oleB (pOR501) and oleC (pOR800). oleA and oleC are closely linked in the chromosome as they were both obtained together in two cosmid clones that were isolated from a genomic library. Sequencing of the oleC resistance determinant revealed four complete open reading frames (ORFs) and the C-terminal end of a fifth. The functions of orf1 and orf2 are unknown since they did not show significant similarity with other sequences in the data bases. The orf3 gene product has similarity with some proteins involved in iron and vitamin B₁₂ uptake in bacteria. The orf4 gene product had a hydrophilic profile and showed important similarity with proteins containing typical ATP-binding domains characteristic of the ABC-transporter superfamily and involved in membrane transport and, particularly, with several genes conferring resistance to various macrolide antibiotics and anticancer drugs. The last gene, orf5, is translationally coupled to orf4 and codes for a hydrophobic polypeptide containing several trans-membrane domains characteristic of integral membrane proteins. Subcloning and deletion experiments limited the resistance determinant to a 0.9kb Pst1-Sph1 fragment and only orf4 is included in this fragment. These results suggest that resistance to oleandomycin conferred by oleC (orf4) is probably due to an efflux transport system of the ABC-transporter superfamily.     

Homologous potyvirus and flavivirus proteins belonging to a superfamily of helicase-like proteins

Comparison of the nucleoside triphosphate-binding motif(NTBM)-containing proteins of two groups of apparently distantly related positive-strand RNA viruses (potyvirus and flavivirus), revealed significant sequence similarity. In addition, these two groups of viral proteins show amino acid motifs in common with those conserved in a group of five NTBM-containing proteins from prokaryotic and eukaryotic cells, some of which have been experimentally related to helicase activity. Here we propose that the proteins mentioned above constitute a superfamily of helicase-like proteins, distinct from the one previously described [Gorbalenya et al., FEBS Lett. 235 (1988) 16-24; Hodgman, Nature 333 (1988) 22-23; 578], which includes the NTBM-containing proteins from another group of positive-strand RNA viruses, the 'Sindbis-like' viruses.     

Characterization of a novel hydroxynitrile lyase from Nandina domestica Thunb

The leaves of Nandina domestica Thunb. exhibited high hydroxynitrile lyase (HNL) activity in (R)-mandelonitrile synthesis. The specific activity of young leaves was significantly higher than that of mature leaves. We isolated two HNLs with molecular mass of 24.9 kDa (NdHNL-S) and 28.0 kDa (NdHNL-L) from the young leaves. Both NdHNLs were composed of two identical subunits, without FAD and carbohydrates. We purified NdHNL-L and revealed its enzymatic properties. The whole deduced amino acid sequence of NdHNL-L was not homologous to any other HNLs, and the specific activity for mandelonitrile synthesis by NdHNL-L was higher than that by other plant HNLs. The enzyme catalyzed enantioselective synthesis of (R)-cyanohydrins, exhibited high activity at pH 4.0, and high stability in the pH range of 3.5–8.0 and below 55°C. Thus, NdHNL-L is a novel HNL with novel amino acid sequence and has a potential for the efficient production of (R)-cyanohydrins.