Streptomyces glucose/xylose isomerase has a single active site for glucose and xylose

A kinetic method which allows one to evaluate whether an enzyme acting on two different substrates has one or two active sites was employed to study the active site of glucose isomerase which catalyses the isomerization of both glucose and xylose. The experimental data on the rates of hydrolysis of mixtures of various concentrations of glucose and xylose by the glucose isomerase from Streptomyces coincides well with the theoretical values calculated for the case of a single active site.     

Evidence for two distinct active sites on aldehyde dehydrogenase

Aldehyde dehydrogenase can catalyze the hydrolysis of esters such as p-nitrophenyl acetate as well as oxidize aldehydes to acids. It has not been proven unequivocally that the two reactions occur at the same active site. In the accompanying paper (Tu, G. C., and Weiner, H. (1988) J. Biol. Chem. 263, 1212-1217) evidence was presented which showed that cysteine at position 49 was at the active site for the dehydrogenase reaction. Evidence also was presented which showed that cysteine located at position 162 was susceptible to modification by N-ethylmaleimide. It was shown here that the two activities of the enzyme can be differently protected from inactivation by substrate analogs. Furthermore, aldehydes were found to be poor inhibitors against the esterase reaction while ester was a good inhibitor against the dehydrogenase reaction. In addition, it was possible to modify cysteine 49 with N-ethylmaleimide but not find inhibition of the esterase reactivity until cysteine 162 was modified. It appears that horse liver aldehyde dehydrogenase has two separate active sites per subunit. The data fit a model where ester can be hydrolyzed at both sites but that aldehyde oxidation occurred only at position 49.     

The monomeric homing endonuclease PI-SceI has two catalytic centres for cleavage of the two strands of its DNA substrate

The monomeric homing endonuclease PI-SceI cleaves the two strands of its DNA substrate in a concerted manner, which raises the question of whether this enzyme harbours one or two catalytic centres. If PI-SceI has only one catalytic centre, one would expect that cross-linking enzyme and substrate should prevent reorientation of the enzyme required to perform the second cut after having made the first cut: PI-SceI, however, when cross-linked to its substrate, is able to cleave both DNA strands. If PI-SceI has two catalytic centres, one would expect that it should be possible to inactivate one catalytic centre by mutation and obtain a variant with preference for a substrate nicked in one strand; such variants have been found. The structural homology between the catalytic domain of PI-SceI having a pseudo 2-fold symmetry, and I-CreI, a homodimeric homing endonuclease, suggests that in PI-SceI active site I, which attacks the top strand, comprises Asp218, Asp229 and Lys403, while Asp326, Thr341 and Lys301 make up active site II, which cleaves the bottom strand. Cleavage experiments with modified oligodeoxynucleotides and metal ion mapping experiments demonstrate that PI-SceI interacts differently with the two strands at the cleavage position, supporting a model of two catalytic centres.     

Malonyl-CoA:ACP transacylase from Streptomyces coelicolor has two alternative catalytically active nucleophiles

Fatty acids and polyketides are synthesized by mechanistically and evolutionarily related multienzyme systems. Their carbon chain backbones are synthesized via repeated decarboxylative condensations of alpha-carboxylated building blocks onto a growing acyl chain. These alpha-carboxylated building blocks are transferred from the corresponding coenzyme A thioesters onto the phosphopantetheine arm of an acyl carrier protein (ACP) by acyl transferases, which operate by a ping-pong mechanism involving an acyl-O-serine intermediate. In the course of our studies on the malonyl-CoA:ACP transacylase (MAT) from Streptomyces coelicolor, we observed that an active-site Ser (97) --> Ala mutant retains activity as well as the ability to be covalently labeled by (14)C malonyl-CoA. Here we demonstrate that an alternative, catalytically competent nucleophile exists in the active site of this enzyme. Next to the active-site serine is a histidine residue that is conserved in some, but not all acyl transferases. The H96A mutant is also active and can be labeled, but an H96A/S97A double mutant is inactive and cannot be labeled. The ability of H96 to form a malonyl-imidazole adduct was confirmed by proteolysis, followed by radio-HPLC and mass spectrometric analysis of the S97A mutant enzyme. Kinetic analysis revealed that the k(cat) of the S97A mutant was within 10-fold that of the wild-type enzyme, whereas the K(M)s of the two enzymes were comparable. Sequence comparison with the E. coli MAT (whose X-ray structure is known) led to the identification of H201 as the putative base in the serine-histidine catalytic dyad of the S. coelicolor enzyme. The absence of MAT activity in the H201A mutant and the detection of weak activity in the H201Q mutant was consistent with this proposal. The implications of this unexpected finding are discussed.     

Evidence for two catalytically active kinase domains in pp90rsk.

Mitogen-activated protein kinase and one of its targets, pp90rsk (ribosomal S6 kinase [RSK]), represent two serine/threonine kinases in the Ras-activated signalling cascade that are capable of directly regulating gene expression. pp90rsk has been shown to have two highly conserved and distinct catalytic domains. However, whether both domains are active and which domain is responsible for its various identified phosphotransferase activities have not been determined. Here we demonstrate that the N-terminal domain is responsible for its phosphotransferase activity towards a variety of substrates which contain an RXXS motif at the site of in vitro phosphorylation, including serum response factor, c-Fos, Nur77, and the 40S ribosomal protein S6. We also provide evidence that the C-terminal domain is catalytically active and can be further activated by mitogen-activated protein kinase phosphorylation.     

Evidence for sequential action of two ATPase active sites in yeast Msh2-Msh6

Bacterial MutS homodimers contain two ATPase active sites that have non-equivalent functions in DNA mismatch repair. The homologous Msh2-Msh6 complex in eukaryotes also has intrinsic ATPase activity that is essential for mismatch repair. Here, we investigate differences in the two putative ATPase active sites by examining the properties of heterodimers containing alanine substituted for an invariant glutamic acid in the active site of either Msh2, Msh6 or both. Mutation rates in wild type versus Glu-->Ala mutant haploid yeast strains indicate that both ATPase active sites are essential for mismatch repair activity in vivo. The properties of purified heterodimers suggest that the ATPase active site in Msh6 binds ATP with higher affinity and hydrolyzes ATP faster and with higher efficiency than does the ATPase active site in Msh2. This suggests sequential action of the two ATPase active sites, in which ATP binds to Msh6 first to trigger downstream events in mismatch repair.     

Yeast glyoxalase I is a monomeric enzyme with two active sites

The tertiary structure of the monomeric yeast glyoxalase I has been modeled based on the crystal structure of the dimeric human glyoxalase I and a sequence alignment of the two enzymes. The model suggests that yeast glyoxalase I has two active sites contained in a single polypeptide. To investigate this, a recombinant expression clone of yeast glyoxalase I was constructed for overproduction of the enzyme in Escherichia coli. Each putative active site was inactivated by site-directed mutagenesis. According to the alignment, glutamate 163 and glutamate 318 in yeast glyoxalase I correspond to glutamate 172 in human glyoxalase I, a Zn(II) ligand and proposed general base in the catalytic mechanism. The residues were each replaced by glutamine and a double mutant containing both mutations was also constructed. Steady-state kinetics and metal analyses of the recombinant enzymes corroborate that yeast glyoxalase I has two functional active sites. The activities of the catalytic sites seem to be somewhat different. The metal ions bound in the active sites are probably one Fe(II) and one Zn(II), but Mn(II) may replace Zn(II). Yeast glyoxalase I appears to be one of the few enzymes that are present as a single polypeptide with two active sites that catalyze the same reaction.     

Scooter, a new active transposon in Schizophyllum commune, has disrupted two genes regulating signal transduction

Two copies of scooter, a DNA-mediated transposon in the basidiomycetous fungus Schizophyllum commune, were characterized. Scooter is the first transposon isolated from S. commune. Scooter creates 8-bp target site duplications, comparable to members of the hAT superfamily, and has 32-bp terminal inverted repeats. Both copies of scooter are nonautonomous elements capable of movement. Southern blot hybridizations show that scooter-related sequences are present in all S. commune strains tested. Scooter-1 was identified initially as an insertion in the Bbeta2 pheromone receptor gene, bbr2, leading to a partial defect in mating. Scooter-2 spontaneously disrupted a gene to produce the frequently occurring morphological mutant phenotype known as thin. The scooter-2 insert permitted cloning of the disrupted gene, thn1, which encodes a putative regulator of G protein signaling (RGS) protein. Spontaneous insertion of scooter into genes with identifiable mutant phenotypes constitutes the first evidence of active transposition of a DNA-mediated transposon in a basidiomycete.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli. 2. Evidence for two different active sites

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase by substrate analogues, by the end product, tyrosine, and by the protein modifying agent iodoacetate has been investigated. The purpose of the investigations was to determine if the two reactions catalyzed by the enzyme occur at a single active site or at two separate active sites. Evidence in support of the conclusion that the mutase and dehydrogenase reactions are catalyzed at two similar but distinct active sites comes from the following results: (1) A substrate analogue (endo-oxabicyclic diacid) that inhibits competitively the mutase reaction has no effect on the dehydrogenase reaction. (2) Malonic acid and several of its derivatives act as inhibitory analogues of chorismate in the mutase reaction and of prephenate in the dehydrogenase reaction. However, different dissociation constants for their interaction with the free enzyme are obtained from studies on the mutase and dehydrogenase reactions. (3) The kinetics of the inhibition by tyrosine of the mutase reaction in the presence of NAD differ from those of the dehydrogenase reaction. The results confirm that carboxymethylation with iodoacetate of one cysteine residue per subunit eliminates both mutase and dehydrogenase activities and show that the inactivation of the enzyme activities is due to iodoacetate functioning as an active site directed inhibitor.     

The receptor-like protein tyrosine phosphatase HPTP alpha has two active catalytic domains with distinct substrate specificities.

Cloning and expression of the homologous domains of the receptor-like tyrosine phosphatase HPTP alpha shows that both domain 1 (D1) and domain 2 (D2) are enzymatically active. The two domains display different substrate specificities with D1 preferentially dephosphorylating MBP approximately RR-src greater than PNPP while D2 favours PNPP much much greater than RR-src and is inactive towards MBP. Each domain has lower activity than an expressed protein containing both domains. Analysis of chimaeric D1/2 proteins suggests that no particular region of D2 is responsible for the low activity of D2 on RR-src and that the specificity differences of D1 and D2 reflect overall sequence dissimilarities. Activities of D1 and D2 are inhibited by zinc, vanadate and EDTA and differentially susceptible to inhibition by heparin and poly(Glu4:Tyr1). Unusually, the activity of the protein containing both domains is stimulated by these polyanions. Regions amino-terminal to each domain are important for catalysis since deletion of these sequences abolishes phosphatase activity. Activity of the double domain polypeptide was also lost upon deletion of the sequence amino-terminal to D1, indicating that inactivation of D1 may suppress D2 activity. Differences in substrate specificity and responses to effectors and the interdependence between the two domains are likely important properties in the function of this PTPase in signal transduction.     

SpoIVB has two distinct functions during spore formation in Bacillus subtilis

The Bacillus subtilis SpoIVB protein is a critical component of the intercompartmental signal-transduction pathway that activates the sigma factor, σ^K, in the mother cell of the sporulating cell. SpoIVB, synthesized in the forespore chamber, must act across two layers of phospholipid membrane to facilitate proteolytic processing of inactive pro-σ^K to active σ^K. We have used a genetic approach to dissect SpoIVB function and found that this protein has two distinct developmental functions. One function is that of intercompartmental signalling of pro-σ^K processing. The other role is essential to spore formation and is illustrated by mutations of SpoIVB which allow cell–cell signalling of pro-σ^K processing but prevent the formation of viable spores. Using localized and site-specific mutagenesis we have identified a functional domain of SpoIVB that is involved in its non-signalling role.     

Engineering Pichia pastoris for biocatalysis: co-production of two active enzymes

High levels of active glycolate oxidase from spinach (GO) and active catalase T from Saccharomyces cerevisiae (catT) have been co-produced in the methylotrophic yeast Pichia pastoris (Pp). In sequential rounds of transformation using two selectable markers, multiple copies of the genes encoding GO and catT were integrated into the Pp chromosome under control of the methanol inducible alcohol oxidase I promoter, resulting in a strain designated MSP8.6. MSP8.6 is a second-generation biocatalyst used for the conversion of glycolate to glyoxylate in the presence of a reaction component which inhibits endogenous Pp catalase. This work demonstrates a significant advance in the utility of recombinant Pp for commercial bioprocess development.     

An unusual tandem-domain rhodanese harbouring two active sites identified in Desulfitobacterium hafniense

The rhodanese protein domain is common throughout all kingdoms of life and is characterized by an active site cysteine residue that is able to bind sulfane sulfur and catalyse sulfur transfer. No unique function has been attributed to rhodanese-domain-containing proteins, most probably because of their diversity at both the level of sequence and protein domain architecture. In this study, we investigated the biochemical properties of an unusual rhodanese protein, PhsE, from Desulfitobacterium?hafniense strain TCE1 which we have previously shown to be massively expressed under anaerobic respiration with tetrachloroethene. The peculiarity of the PhsE protein is its domain architecture which is constituted of two rhodanese domains each with an active site cysteine. The N-terminal rhodanese domain is preceded by a lipoprotein signal peptide anchoring PhsE on the outside of the cytoplasmic membrane. In?vitro sulfur-transferase activity of recombinant PhsE variants was measured for both domains contrasting with other tandem-domain rhodaneses in which usually only the C-terminal domain has been found to be active. The genetic context of phsE shows that it is part of a six-gene operon displaying homology with gene clusters encoding respiratory molybdoenzymes of the PhsA/PsrA family, possibly involved in the reduction of sulfur compounds. Our data suggest, however, that the presence of sulfide in the medium is responsible for the high expression of PhsE in Desulfitobacterium, where it could play a role in the sulfur homeostasis of the cell.     

Reconstitution of active octameric mitochondrial creatine kinase from two genetically engineered fragments.

Creatine kinase (CK) has been postulated to consist of two flexibly hinged domains. A previously demonstrated protease-sensitive site in M-CK (Morris & Jackson, 1991) has directed our attempts to dissect mitochondrial CK (Mi-CK) into two protein fragments encompassing amino acids [1-167] and [168-380]. When expressed separately in Escherichia coli, the two fragments yielded large amounts of insoluble inclusion bodies, from which the respective polypeptides could be purified by a simple two-step procedure. In contrast, co-expression of the two fragments yielded a soluble, active, and correctly oligomerizing enzyme. This discontinuous CK showed nearly full specific activity and was virtually indistinguishable from native Mi-CK by far- and near-UV CD. However, the positive cooperativity of substrate binding was abolished, suggesting a role of the covalent domain linkage in the crosstalk between the substrate binding sites for ATP and creatine. The isolated C-terminal fragment refolded into a native-like conformation in vitro, whereas the N-terminal fragment was largely unfolded. Prefolded [168-380] interacted in vitro with [1-167] to form an active enzyme. Kinetic analysis indicated that the fragments associate rapidly and with high affinity (1/K1 = 17 microM) and then isomerize slowly to an active enzyme (k2 = 0.12 min-1; k-2 = 0.03 min-1). Our data suggest that the C-terminal fragment of Mi-CK represents an autonomous folding unit, and that the folding of the C-terminal part might precede the conformational stabilization of the N-terminal moiety in vivo.     

Paramyxovirus F1 protein has two fusion peptides: implications for the mechanism of membrane fusion

Viral fusion proteins contain a highly hydrophobic segment, named the fusion peptide, which is thought to be responsible for the merging of the cellular and viral membranes. Paramyxoviruses are believed to contain a single fusion peptide at the N terminus of the F1 protein. However, here we identified an additional internal segment in the Sendai virus F1 protein (amino acids 214-226) highly homologous to the fusion peptides of HIV-1 and RSV. A synthetic peptide, which includes this region, was found to induce membrane fusion of large unilamellar vesicles, at concentrations where the known N-terminal fusion peptide is not effective. A scrambled peptide as well as several peptides from other regions of the F1 protein, which strongly bind to membranes, are not fusogenic. The functional and structural characterization of this active segment suggest that the F1 protein has an additional internal fusion peptide that could participate in the actual fusion event. The presence of homologous regions in other members of the same family suggests that the concerted action of two fusion peptides, one N-terminal and the other internal, is a general feature of paramyxoviruses.     

Rainbow trout has two genes for growth hormone

We report the primary structures of two mRNA species (GH1 and GH2), each predicted from the cloned cDNA and genomic gene sequences, that encode growth hormone in rainbow trout (Salmo gairdneri). Both GH1 and GH2 mRNA contain open reading frames comprising 630 nucleotides and encode 210 amino acid residues, of which 11 are variant. The translated regions of mRNA are flanked by a short 5'-untranslated sequence, which is highly conserved, and a relatively long 3'-untranslated sequence, which is highly divergent. The differences at the 3'-untranslated regions suggest that the GH1 and GH2 mRNA originate from different loci. RNA blot analysis of trout pituitary RNA using an oligonucleotide probe specific for the GH2 sequence indicates that the cloned gene is expressed. The GH1 and GH2 mRNA likely are transcribed from two distinct loci, which were duplicated during tetraploidization of the salmonid genome between 50 and 100 million years ago.     

Ceruloplasmin has a distinct active site for the catalyzing glutathione-dependent reduction of alkyl hydroperoxide.

Ceruloplasmin, a blue multi-copper alpha(2)-glycoprotein found in the plasma of all vertebrates, is capable of oxidizing aromatic amines and ferrous iron. Here, we report that human ceruloplasmin exhibits an alkyl hydroperoxide peroxidase activity, which is independent of the oxidase activity. The site-specific modification of the sulfhydryl of cysteine at position 699 in ceruloplasmin completely abolished the antioxidant activity, suggesting that ceruloplasmin is a peroxidase with a cysteinyl thiol as a functional nucleophile. The crystal structure of human ceruloplasmin reveals that the domain containing Cys-699 is apart from the multi-copper complex domains. Taken together, these data suggest that ceruloplasmin has a distinct active site for a glutathione-linked peroxidase activity apart from the copper complex site exerting ferroxidase activity.     

Dynamics comparison of two myoglobins with a distinct heme active site

Sperm whale myoglobin (swMb) is a well-studied heme protein, both experimentally and theoretically. Comparatively, little attention has been paid to another member of Mb family, Aplysia limacina myoglobin (apMb). swMb and apMb have the same overall structure and perform the same biological function, i.e., O₂ carrier, while using a distinct heme active site. To provide insights into the structure-function relationship for these two Mbs, we herein made a comparison in terms of their dynamics properties using molecular dynamics simulation. We analyzed the overall structure and protein motions, as well as the intramolecular contacts, namely salt-bridges and hydrogen bonds, especially the interactions between the heme propionate groups and the polypeptide chain. The internal cavities in apMb were also compared to the well-known xenon and other cavities in swMb. Based on current simulations, we propose a unique “arginine gate” for apMb, which has a similar function to the histidine gate observed for swMb in previous studies. This study provides valuable information for understanding the homology of heme proteins, and also aids in rational design of structural and functional heme proteins by alternating the heme active site.     

Identical behavior of the two active sites of myosin with respect to trinitrophenylation.

Myosin and its active subfragments were trinitrophenylated under conditions in which mainly the active site(s) was modified. Proteins modified at the active site(s) could be separated by affinity chromatography on agarose-ATP columns. By two independent methods, ATPase activity measurements and analysis of elution patterns on agarose-ATP columns, it was shown that the introduction of two trinitrophenyl groups per myosin or one per heavy meromyosin subfragment 1 molecule is responsible for the remarkable change in the ATPase activities. Heavy meromyosin subfragment 1 prepared from trinitrophenylated myosin retained the original degree of trinitrophenylation per "active head." The kinetic constant of trinitrophenylation of the epsilon-amino group of lysine at the active site was found to be 2000 S-1-M-1, whereas a much smaller constant of 2.2 S-1-M-1 was obtained for the trinitrophenylation of the unessential lysyl residues of myosin. By using affinity chromatography, we could follow the formation of mono- and ditrinitrophenyl myosin. The amounts of these myosin derivatives at various extents of the reaction corresponded approximately to the calculated amounts, assuming a random and independent trinitrophenylation of the two myosin "heads." It is concluded that in each of the two heads of myosin there is one ATPase active site and these two sites behave in an identical manner with respect to trinitrophenylation.     

The two homologous domains of human angiotensin I-converting enzyme are both catalytically active.

Molecular cloning of human endothelial angiotensin I-converting enzyme (kininase II; EC 3.4.15.1) (ACE) has recently shown that the enzyme contains two large homologous domains (called here the N and C domains), each bearing a putative active site, identified by sequence comparisons with the active sites of other zinc metallopeptidases. However, the previous experiments with zinc or competitive ACE inhibitors suggested a single active site in ACE. To establish whether both domains of ACE are enzymatically active, a series of ACE mutants, each containing only one intact domain, were constructed by deletion or point mutations of putative critical residues of the other domain, and expressed in heterologous Chinese hamster ovary cells. Both domains are enzymatically active and cleave the C-terminal dipeptide of hippuryl-His-Leu or angiotensin I. Moreover, both domains have an absolute zinc requirement for activity, are activated by chloride and are sensitive to competitive ACE inhibitors, and appear to function independently. However, the two domains display different catalytic constants and different patterns of chloride activation. At high chloride concentrations, the C domain hydrolyzes the two substrates tested faster than does the N domain. His-361,365 and His-959,963 are established as essential residues in the N and C domains, respectively, most likely involved in zinc binding, and Glu-362 in the N domain and Glu-960 in the C domain are essential catalytic residues. These observations provide strong evidence that ACE possesses two independent catalytic domains and suggest that they may have different functions.