Substitution of glutamate-3, valine-19, leucine-23, and serine-24 with alanine in the N-terminal region of human heart muscle carnitine palmitoyltransferase I abolishes malonyl CoA inhibition and binding

The muscle isoform of carnitine palmitoyltransferase I (M-CPTI) is 30- to 100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). We have previously shown that deletion of the first 28 N-terminal amino acid residues in M-CPTI abolished malonyl CoA inhibition and high-affinity binding [Biochemistry 39 (2000) 712-717]. To determine the role of specific residues within the first 28 N-terminal amino acids of human heart M-CPTI on malonyl CoA sensitivity and binding, we constructed a series of substitution mutations and a mutant M-CPTI composed of deletion 18 combined with substitution mutations V19A, L23A, and S24A. All mutants had CPT activity similar to that of the wild type. A change of Glu3 to Ala resulted in a 60-fold decrease in malonyl CoA sensitivity and loss of high-affinity malonyl CoA binding. A change of His5 to Ala in M-CPTI resulted in only a 2-fold decrease in malonyl CoA sensitivity and a significant loss in the low- but not high-affinity malonyl CoA binding. Deletion of the first 18 N-terminal residues combined with substitution mutations V19A, L23A, and S24A resulted in a mutant M-CPTI with an over 140-fold decrease in malonyl CoA sensitivity and a significant loss in both high- and low-affinity malonyl CoA binding. This was further confirmed by a combined four-residue substitution of Glu3, Val19, Leu23, and Ser24 with alanine. Our site-directed mutagenesis studies demonstrate that Glu3, Val19, Leu23, and Ser24 in M-CPTI are important for malonyl CoA inhibition and binding, but not for catalysis.     

The first 28 N-terminal amino acid residues of human heart muscle carnitine palmitoyltransferase I are essential for malonyl CoA sensitivity and high-affinity binding

Heart/skeletal muscle carnitine palmitoyltransferase I (M-CPTI) is 30-100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). To determine the role of the N-terminal region of human heart M-CPTI on malonyl CoA sensitivity and binding, a series of deletion mutations were constructed ranging in size from 18 to 83 N-terminal residues. All of the deletions except Delta83 were active. Mitochondria from the yeast strains expressing Delta28 and Delta39 exhibited a 2.5-fold higher activity compared to the wild type, but were insensitive to malonyl CoA inhibition and had complete loss of high-affinity malonyl CoA binding. The high-affinity site (K(D1), B(max1)) for binding of malonyl CoA to M-CPTI was completely abolished in the Delta28, Delta39, Delta51, and Delta72 mutants, suggesting that the decrease in malonyl CoA sensitivity observed in these mutants was due to the loss of the high-affinity binding entity of the enzyme. Delta18 showed only a 4-fold loss in malonyl CoA sensitivity but had activity and high-affinity malonyl CoA binding similar to the wild type. Replacement of the N-terminal domain of L-CPTI with the N-terminal domain of M-CPTI does not change the malonyl CoA sensitivity of the chimeric L-CPTI, suggesting that the amino acid residues responsible for the differing sensitivity to malonyl CoA are not located in this N-terminal region. These results demonstrate that the N-terminal residues critical for activity and malonyl CoA sensitivity in M-CPTI are different from those of L-CPTI.     

Deletion mutagenesis of human cystathionine beta-synthase. Impact on activity,oligomeric status,and S-adenosylmethionine regulation

Cystathionine beta-synthase is a tetrameric hemeprotein that catalyzes the pyridoxal 5'-phosphate-dependent condensation of serine and homocysteine to cystathionine. We have used deletion mutagenesis of both the N and C termini to investigate the functional organization of the catalytic and regulatory regions of this enzyme. Western blot analysis of these mutants expressed in Escherichia coli indicated that residues 497-543 are involved in tetramer formation. Deletion of the 70 N-terminal residues resulted in a heme-free protein retaining 20% of wild type activity. Additional deletion of 151 C-terminal residues from this mutant resulted in an inactive enzyme. Expression of this double-deletion mutant as a glutathione S-transferase fusion protein generated catalytically active protein (15% of wild type activity) that was unaffected by subsequent removal of the fusion partner. The function of the N-terminal region appears to be primarily steric in nature and involved in the correct folding of the enzyme. The C-terminal region of human cystathionine beta-synthase contains two hydrophobic motifs designated "CBS domains." Partial deletion of the most C-terminal of these domains decreased activity and caused enzyme aggregation and instability. Removal of both of these domains resulted in stable constitutively activated enzyme. Deletion of as few as 8 C-terminal residues increased enzyme activity and abolished any further activation by S-adenosylmethionine indicating that the autoinhibitory role of the C-terminal region is not exclusively a function of the CBS domains.     

Cysteine-scanning mutagenesis of muscle carnitine palmitoyltransferase I reveals a single cysteine residue (Cys-305) is important for catalysis

Carnitine palmitoyltransferase (CPT) I catalyzes the conversion of long-chain fatty acyl-CoAs to acyl carnitines in the presence of l-carnitine, a rate-limiting step in the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix. To determine the role of the 15 cysteine residues in the heart/skeletal muscle isoform of CPTI (M-CPTI) on catalytic activity and malonyl-CoA sensitivity, we constructed a 6-residue N-terminal, a 9-residue C-terminal, and a 15-residue cysteineless M-CPTI by cysteine-scanning mutagenesis. Both the 9-residue C-terminal mutant enzyme and the complete 15-residue cysteineless mutant enzyme are inactive but that the 6-residue N-terminal cysteineless mutant enzyme had activity and malonyl-CoA sensitivity similar to those of wild-type M-CPTI. Mutation of each of the 9 C-terminal cysteines to alanine or serine identified a single residue, Cys-305, to be important for catalysis. Substitution of Cys-305 with Ala in the wild-type enzyme inactivated M-CPTI, and a single change of Ala-305 to Cys in the 9-residue C-terminal cysteineless mutant resulted in an 8-residue C-terminal cysteineless mutant enzyme that had activity and malonyl-CoA sensitivity similar to those of the wild type, suggesting that Cys-305 is the residue involved in catalysis. Sequence alignments of CPTI with the acyltransferase family of enzymes in the GenBank led to the identification of a putative catalytic triad in CPTI consisting of residues Cys-305, Asp-454, and His-473. Based on the mutagenesis and substrate labeling studies, we propose a mechanism for the acyltransferase activity of CPTI that uses a catalytic triad composed of Cys-305, His-473, and Asp-454 with Cys-305 serving as a probable nucleophile, thus acting as a site for covalent attachment of the acyl molecule and formation of a stable acyl-enzyme intermediate. This would in turn allow carnitine to act as a second nucleophile and complete the acyl transfer reaction.     

The carboxyl terminus of coffee bean alpha-galactosidase is critical for enzyme activity

The role of the carboxyl (C)-terminal region of coffee bean alpha-galactosidase (alpha-GAL) has been studied by expressing C-terminal deletion mutants in the methylotrophic yeast strain Pichia pastoris. A previous study of human alpha-galactosidase determined that enzyme activity increased when up to 10 amino acid residues were deleted. Deleting 11 residues reduced activity, and deleting 12 residues abolished activity. In our studies, alpha-GAL activity is reduced when one or two amino acids are deleted, as is enzyme secretion directed by P. pastoris signal sequences. The pH profile is similar to that of the wild-type enzyme. Deleting 3 or more residues from the C-terminal end results in a complete loss of both enzyme secretion and activity. The C-terminus of alpha-GAL seems to play an important role in overall enzyme conformation and may directly affect the proper conformation of the active site.     

Role of C-terminal region of Staphylococcal nuclease for foldability,stability, and activity

The role of the C-terminal region of Staphylococcal nuclease (SNase) was examined by deletion mutation. Deletions up to eight residues do not affect the structure and function. The structure and enzymatic activity were partially lost by deleting Ser141-Asn149 (Delta141-149), and deletion of Trp140-Asn149 (Delta140-149) resulted in further loss of structure and activity. A 13-residue deletion showed the same effect as the 10-residue deletion. Both Ser141Gln and Ser141Ala mutations for an eight-residue deletion mutant did not alter properties as well as Ser141A1a for full-length SNase. In contrast, Trp140Ala mutation for Delta141-149 shows the same effect as the deletion of Trp140. Trp140Ala mutation for full-length SNase causes the loss of native structure. These observations indicate the significance of the 140th and the 141st residues. The side-chain of the 140th residue is required to be tryptophan; however, the backbone of the 141st residue is solely critical for foldability, but the side-chain information is not crucial. All of the mutants that take a non-native conformation show enzymatic activity and inhibitor-induced folding, suggesting that foldability is required for the activity.     

Disrupted plasma membrane localization and loss of function reveal regions of human equilibrative nucleoside transporter 1 involved in structural integrity and activity

Human Equilibrative Nucleoside Transporter 1 (hENT1) is an integral membrane protein that transports nucleosides and analog drugs across cellular membranes. Very little is known about intracellular processing and localization of hENT1. Here we show that disruption of a highly conserved triplet (PWN) near the N-terminus, or the last eight C-terminal residues (two hydrophobic triplets separated by a positive arginine) result in loss of plasma membrane localization and/or transport function. To understand the role of specific residues within these regions, we studied the localization patterns of N- or C-terminal deletion and/or substitution mutants of GFP-hENT1 using confocal microscopy. Quantification of GFP-hENT1 (mutant and wildtype) protein at the plasma membrane was conducted using nitrobenzylthioinosine (NBTI) binding. Functionality of the GFP-hENT1 mutants was determined by heterologous expression in Xenopus laevis oocytes followed by measurement of uridine uptake. Mutation of the proline within the PWN motif disrupts plasma membrane localization. C-terminal mutations (primarily within the hydrophobic triplets) lead to hENT1 retention within the cell (e.g. in the ER). Some mutants still localize to the plasma membrane but show reduced transport activity. These data suggest that these two regions contribute to the structural integrity and thus correct processing and function of hENT1.     

Expression and characterization of wild type, truncated, and mutant forms of the intracellular region of the receptor-like protein tyrosine phosphatase HPTP beta.

Human HPTP beta is unique among mammalian receptor-like protein tyrosine phosphatases in that it has only a single catalytic domain. The intracellular region of HPTP beta was expressed in bacteria, purified, and characterized. It exhibits high activity toward all substrates tested and is potently inhibited by zinc. Vanadate and polyanions also inhibited activity. The juxta-membrane segment of HPTP beta (residues 1622-1639) potentially functions as a negative regulatory sequence since its deletion can increase HPTP beta activity 5-fold. This segment contains up to two sites for protein kinase C phosphorylation, although in vitro phosphorylation by this kinase did not affect HPTP beta activity. The boundaries of the catalytic domain were delineated by truncation analyses. Successive deletion of N-terminal sequence prior to residue 1684 had little effect on substrate affinity and at most reduced activity about 6-fold. Further removal of residues 1684-1686 resulted in a marked 50-500-fold drop in activity, and loss of N-terminal sequence prior to residue 1690 abolished activity. Based on these analyses a highly conserved motif was identified in all mammalian tyrosine phosphatases (E/q) (F/y)XX(L/i), corresponding to positions 1684-1688 of HPTP beta. Mutation of residue 1684 or 1685 generally gave rise to proteins with marked temperature sensitivity. These mutant HPTP beta were active but had reduced activity compared to the wild type enzyme. In conjunction, these results suggest that this region represents the N-terminal border of the catalytic domain and is essential for correct phosphatase folding although not directly involved in catalysis. Parallel truncation studies have defined residues 1930-1939/40 as the C-terminal border of the catalytic domain.     

Identification of ERK2-binding domain of EBITEIN1, a novel ERK2-binding protein

We recently cloned a cDNA encoding a novel extracellular signal-regulated kinase 2 (ERK2) binding protein, EBITEIN1, by yeast two-hybrid screening. In this study, we further characterized EBITEIN1. Binding experiments using various deletion mutants identified a 40-amino acid minimal sequence for binding ERK2. Binding experiments using substitution mutants indicated the crucial role of arginine residues in this sequence. Based on empirical and bioinformatic analyses, we propose two domains in EBITEIN1. One is the minimal sequence for binding ERK2 (EB domain) and the other is the EBITEIN1 C-terminal domain (ECT domain). These results might pave the way for further empirical and bioinformatic analyses of EBITEIN1- and ERK2-mediated events.     

Mutations in the C-terminal domain of ALSV (Avian Leukemia and Sarcoma Viruses) integrase alter the concerted DNA integration process in vitro.

Integrase (IN) is the retroviral enzyme responsible for the integration of the DNA copy of the retroviral genome into the host cell DNA. The C-terminal domain of IN is involved in DNA binding and enzyme multimerization. We previously performed single amino acid substitutions in the C-terminal domain of the avian leukemia and sarcoma viruses (ALSV) IN. Here, we modelled these IN mutants and analysed their ability to mediate concerted DNA integration (in an in vitro assay) as well as to form dimers (by size exclusion chromatography and protein-protein cross-linking). Mutations of residues located at the dimer interface (V239, L240, Y246, V257 and K266) have the greatest effects on the activity of the IN. Among them: (a) the L240A mutation resulted in a decrease of integration efficiency that was concomitant with a decrease of IN dimerization; (b) the V239A, V249A and K266A mutants preferentially mediated non-concerted DNA integration rather than concerted DNA integration although they were found as dimers. Other mutations (V260E and Y246W/DeltaC25) highlight the role of the C-terminal domain in the general folding of the enzyme and, hence, on its activity. This study points to the important role of residues at the IN C-terminal domain in the folding and dimerization of the enzyme as well as in the concerted DNA integration of viral DNA ends.     

The last 10 amino acid residues beyond the hydrophobic motif are critical for the catalytic competence and function of protein kinase Calpha

The segment C-terminal to the hydrophobic motif at the V5 domain of protein kinase C (PKC) is the least conserved both in length and in amino acid identity among all PKC isozymes. By generating serial truncation mutants followed by biochemical and functional analyses, we show here that the very C terminus of PKCalpha is critical in conferring the full catalytic competence to the kinase and for transducing signals in cells. Deletion of one C-terminal amino acid residue caused the loss of approximately 60% of the catalytic activity of the mutant PKCalpha, whereas deletion of 10 C-terminal amino acid residues abrogated the catalytic activity of PKCalpha in immune complex kinase assays. The PKCalpha C-terminal truncation mutants were found to lose their ability to activate mitogen-activated protein kinase, to rescue apoptosis induced by the inhibition of endogenous PKC in COS cells, and to augment melatonin-stimulated neurite outgrowth. Furthermore, molecular dynamics simulations revealed that the deletion of 1 or 10 C-terminal residues results in the deformation of the V5 domain and the ATP-binding pocket, respectively. Finally, PKCalpha immunoprecipitated using an antibody against its C terminus had only marginal catalytic activity compared with that of the PKCalpha immunoprecipitated by an antibody against its N terminus. Therefore, the very C-terminal tail of PKCalpha is a novel determinant of the catalytic activity of PKC and a promising target for selective modulation of PKCalpha function. Molecules that bind preferentially to the very C terminus of distinct PKC isozymes and suppress their catalytic activity may constitute a new class of selective inhibitors of PKC.     

Insights into the maturation of hyperthermophilic pyrolysin and the roles of its N-terminal propeptide and long C-terminal extension

Pyrolysin-like proteases from hyperthermophiles are characterized by large insertions and long C-terminal extensions (CTEs). However, little is known about the roles of these extra structural elements or the maturation of these enzymes. Here, the recombinant proform of Pyrococcus furiosus pyrolysin (Pls) and several N- and C-terminal deletion mutants were successfully expressed in Escherichia coli. Pls was converted to mature enzyme (mPls) at high temperatures via autoprocessing of both the N-terminal propeptide and the C-terminal portion of the long CTE, indicating that the long CTE actually consists of the C-terminal propeptide and the C-terminal extension (CTEm), which remains attached to the catalytic domain in the mature enzyme. Although the N-terminal propeptide deletion mutant PlsΔN displayed weak activity, this mutant was highly susceptible to autoproteolysis and/or thermogenic hydrolysis. The N-terminal propeptide acts as an intramolecular chaperone to assist the folding of pyrolysin into its thermostable conformation. In contrast, the C-terminal propeptide deletion mutant PlsΔC199 was converted to a mature form (mPlsΔC199), which is the same size as but less stable than mPls, suggesting that the C-terminal propeptide is not essential for folding but is important for pyrolysin hyperthermostability. Characterization of the full-length (mPls) and CTEm deletion (mPlsΔC740) mature forms demonstrated that CTEm not only confers additional stability to the enzyme but also improves its catalytic efficiency for both proteineous and small synthetic peptide substrates. Our results may provide important clues about the roles of propeptides and CTEs in the adaptation of hyperthermophilic proteases to hyperthermal environments.     

Conserved amino acids at the C-terminus of rat phospholipase D1 are essential for enzymatic activity.

Rat brain phospholipase D1 (rPLD1) has two highly conserved motifs [H(X)K(X)4D, denoted HKD] located at the N-terminal and C-terminal halves, which are required for activity. Association of the two halves is essential for rPLD1 activity, which probably brings the two HKD domains together to form a catalytic center. In the present study, we find that an intact C-terminus is also essential for the catalytic activity of rPLD1. Serial deletion of the last four amino acids, EVWT, which are conserved in all mammalian PLD isoforms, abolished the catalytic activity of rPLD1. This loss of catalytic activity was not due to a lack of association of the N-terminal and C-terminal halves. Mutations of the last three amino acids showed that substitutions with charged or less hydrophobic amino acids all reduced PLD activity. For example, mutations of Thr1036 and Val1034 to Asp or Lys caused marked inactivation, whereas mutation to other amino acids had less effect. Mutation of Trp1035 to Leu, Ala, His or Tyr caused complete inactivation, whereas mutation of Glu1033 to Ala enhanced activity. The size of the amino acids at the C-terminus also affected the catalytic activity of PLD, reduced activity being observed with conservative mutations within the EVWT sequence (such as T/S, V/L or W/F). The enzyme was also inactivated by the addition of Ala or Val to the C-terminus of this sequence. Interestingly, the inactive C-terminal mutants could be complemented by cotransfection with a wild-type C-terminal half to restore PLD activity in vivo. These data demonstrate that the integrity of the C-terminus of rPLD1 is essential for its catalytic activity. Important features are the hydrophobicity, charge and size of the four conserved C-terminal amino acids. It is proposed that these play important roles in maintaining a functional catalytic structure by interacting with a specific domain within rPLD1.     

The importance of Loop 7 for the activity of calcineurin

Calcineurin (CN) is a heterodimer composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). Loop 7 lies within the CNA catalytic domain. To investigate the role of Loop 7 in enzyme activity, we systematically examined all its residues by site-directed deletion mutation. Our results show that the Loop 7 residues are important for enzyme activity. Besides deleting residues V314, Y315 or N316, enzyme activity also increased dramatically when residues D313 or K318 were deleted. In contrast, almost all activity was lost when L312 or N317 were deleted. Ni2+ and Mn2+ were effective activators for all active mutants. However, whereas the wild-type enzyme was more efficiently activated by Ni2+ than by Mn2+ with 32P-labeled R(II) peptide as substrate, the reverse was true in all the mutants. We also found that the effect of Loop 7 on enzyme activity was substrate dependent, and involved interactions between Loop 7 residues and the unresolved part of the CN crystal structure near the auto-inhibitory domain and catalytic site.     

Functional role of the C terminus of human organic anion transporter hOAT1

Human organic anion transporter hOAT1 plays critical roles in the body disposition of environmental toxins and clinically important drugs. In the present study, we examined the role of the C terminus of hOAT1 in its function. Combined approaches of cell surface biotinylation and transport analysis were employed for such purposes. It was found that deletion of the last 15 amino acids (residues 536-550) or the last 30 amino acids (residues 521-550) had no significant effect on transport activity. However, deletion of the entire C terminus (residues 506-550) completely abolished transport activity. Alanine scanning mutagenesis within the region of amino acids 506-520 led to the discovery of two critical amino acids: Glu-506 and Leu-512. Substitution of negatively charged Glu-506 with neutral amino acids alanine or glutamine resulted in complete loss of transport activity. However, such loss of transport activity could be rescued by substitution of Glu-506 with another negatively charged amino acid aspartic acid, suggesting the importance of negative charge at this position for maintaining the correct tertiary structure of the transporter, possibly by forming a salt bridge with a positively charged amino acid. Substitution of Leu-512 with amino acids carrying progressively smaller side chains including isoleucine, valine, and alanine resulted in mutants (L512I, L512V, and L512A) with increasingly impaired transport activity. However, the cell surface expression of these mutants was not affected. Kinetic analysis of mutant L512V revealed that the reduced transport activity of this mutant resulted mainly from a reduced maximum transport velocity Vmax without affecting the binding affinity (1/Km) of the transporter for its substrates, suggesting that the size of the side chain at position 512 critically affects transporter turnover number. Together, our results are the first to highlight the central role of the C terminus of hOAT1 in the function of this transporter.     

Important role for phylogenetically invariant PP2Acalpha active site and C-terminal residues revealed by mutational analysis in Saccharomyces cerevisiae

PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acalpha functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acalpha Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acalpha catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acalpha C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acalpha catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.     

Identification of the enzymatic active site of tobacco caffeoyl-coenzyme A O-methyltransferase by site-directed mutagenesis.

Animal catechol O-methyltransferases and plant caffeoyl-coenzyme A O-methyltransferases share about 20% sequence identity and display common structural features. The crystallographic structure of rat liver catechol O-methyltransferase was used as a template to construct a homology model for tobacco caffeoyl-coenzyme A O-methyltransferase. Integrating substrate specificity data, the three-dimensional model identified several amino acid residues putatively involved in substrate binding. These residues were mutated by a polymerase chain reaction method and wild-type and mutant enzymes were each expressed in Escherichia coli and purified. Substitution of Arg-220 with Thr resulted in the total loss of enzyme activity, thus indicating that Arg-220 is involved in the electrostatic interaction with the coenzyme A moiety of the substrate. Changes of Asp-58 to Ala and Gln-61 to Ser were shown to increase K(m) values for caffeoyl coenzyme A and to decrease catalytic activity. Deletions of two amino acid sequences specific for plant enzymes abolished activity. The secondary structures of the mutants, as measured by circular dichroism, were essentially unperturbed as compared with the wild type. Similar changes in circular dichroism spectra were observed after addition of caffeoyl coenzyme A to the wild-type enzyme and the substitution mutants but not in the case of deletion mutants, thus revealing the importance of these sequences in substrate-enzyme interactions.     

Sequence-structure-function analysis of the bifunctional enzyme MnmC that catalyses the last two steps in the biosynthesis of hypermodified nucleoside mnm5s2U in tRNA

MnmC catalyses the last two steps in the biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) in tRNA. Previously, we reported that this bifunctional enzyme is encoded by the yfcK open reading frame in the Escherichia coli K12 genome. However, the mechanism of its activity, in particular the potential structural and functional dependence of the domains responsible for catalyzing the two modification reactions, remains unknown. With the aid of the protein fold-recognition method, we constructed a structural model of MnmC in complex with the ligands and target nucleosides and studied the role of individual amino acids and entire domains by site-directed and deletion mutagenesis, respectively. We found out that the N-terminal domain contains residues responsible for binding of the S-adenosylmethionine cofactor and catalyzing the methylation of nm(5)s(2)U to form mnm(5)s(2)U, while the C-terminal domain contains residues responsible for binding of the FAD cofactor. Further, point mutants with compromised activity of either domain can complement each other to restore a fully functional enzyme. Thus, in the conserved fusion protein MnmC, the individual domains retain independence as enzymes. Interestingly, the N-terminal domain is capable of independent folding, while the isolated C-terminal domain is incapable of folding on its own, a situation similar to the one reported recently for the rRNA modification enzyme RsmC.     

GRIM-19 is essential for maintenance of mitochondrial membrane potential

GRIM-19 was found to copurify with complex I of mitochondrial respiratory chain and subsequently was demonstrated to be involved in complex I assembly and activity. To further understand its function in complex I, we dissected its functional domains by generating a number of deletion, truncation, and point mutants. The mitochondrial localization sequences were located at the N-terminus. Strikingly, deletion of residues 70-80, 90-100, or the whole C-terminal region (70-144) led to a loss of mitochondrial transmembrane potential (DeltaPsim). However, similar deletions of another two complex I subunits, NDUFA9 and NDUFS3, did not show such effect. We also found that deletion of the last 10 residues affected GRIM-19's ability to be assembled to complex I. We constructed a dominant-negative mutant containing the N-terminal 60 and the last C-terminal 10 residues, which could be assembled into complex I, but failed to maintain normal DeltaPsim. Cells overexpressing this mutant did not spontaneously undergo cell death, but were sensitized to apoptosis induced by cell death agents. Our results demonstrate that GRIM-19 is required for electron transfer activity of complex I, and disruption of DeltaPsim by GRIM-19 mutants enhances the cells' sensitivity to apoptotic stimuli.     

Structure-function analysis of the human sialyltransferase ST3Gal I: role of n-glycosylation and a novel conserved sialylmotif

All eukaryotic sialyltransferases have in common the presence in their catalytic domain of several conserved peptide regions (sialylmotifs L, S, and VS). Functional analysis of sialylmotifs L and S previously demonstrated their involvement in the binding of donor and acceptor substrates. The region comprised between the sialylmotifs S and VS contains a stretch of four highly conserved residues, with the following consensus sequence (H/y)Y(Y/F/W/h)(E/D/q/g). (Capital letters and lowercase letters indicate a strong or low occurrence of the amino acid, respectively.) The functional importance of these residues and of the conserved residues of motif VS (HX(4)E) was assessed using as a template the human ST3Gal I. Mutational analysis showed that residues His(299) and Tyr(300) of the new motif, and His(316) of the VS motif, are essential for activity since their substitution by alanine yielded inactive enzymes. Our results suggest that the invariant Tyr residue (Tyr(300)) plays an important conformational role mainly attributable to the aromatic ring. In contrast, the mutants W301F, E302Q, and E321Q retained significant enzyme activity (25-80% of the wild type). Kinetic analyses and CDP binding assays showed that none of the mutants tested had any significant effect in nucleotide donor binding. Instead the mutant proteins were affected in their binding to the acceptor and/or demonstrated lower catalytic efficiency. Although the human ST3Gal I has four N-glycan attachment sites in its catalytic domain that are potentially glycosylated, none of them was shown to be necessary for enzyme activity. However, N-glycosylation appears to contribute to the proper folding and trafficking of the enzyme.