A Conserved Histidine Is Essential for Glycerolipid Acyltransferase Catalysis

Sequence analysis of membrane-bound glycerolipid acyltransferases revealed that proteins from the bacterial, plant, and animal kingdoms share a highly conserved domain containing invariant histidine and aspartic acid residues separated by four less conserved residues in an HX₄D configuration. We investigated the role of the invariant histidine residue in acyltransferase catalysis by site-directed mutagenesis of two representative members of this family, the sn-glycerol-3-phosphate acyltransferase (PlsB) and the bifunctional 2-acyl-glycerophosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase (Aas) of Escherichia coli. Both the PlsB[H306A] and Aas[H36A] mutants lacked acyltransferase activity. However, the Aas[H36A] mutant retained significant acyl-acyl carrier protein synthetase activity, illustrating that the lack of acyltransferase activity was specifically associated with the H36A substitution. The invariant aspartic acid residue in the HX₄D pattern was also important. The substitution of aspartic acid 311 with glutamic acid in PlsB resulted in an enzyme with significantly reduced catalytic activity. Substitution of an alanine at this position eliminated acyltransferase activity; however, the PlsB[D311A] mutant protein did not assemble into the membrane, indicating that aspartic acid 311 is also important for the proper folding and membrane insertion of the acyltransferases. These data are consistent with a mechanism for glycerolipid acyltransferase catalysis where the invariant histidine functions as a general base to deprotonate the hydroxyl moiety of the acyl acceptor.     

The conserved asparagine in the HNH motif serves an important structural role in metal finger endonucleases

The HNH motif is a small nucleic acid binding and cleavage module, widespread in metal finger endonucleases in all life kingdoms. Here we studied a non-specific endonuclease, the nuclease domain of ColE7 (N-ColE7), to decipher the role of the conserved asparagine and histidine residues in the HNH motif. We found, using fluorescence resonance energy transfer (FRET) assays, that the DNA hydrolysis activity of H545 N-ColE7 mutants was completely abolished while activities of N560 and H573 mutants varied from 6.9% to 83.2% of the wild-type activity. The crystal structures of three N-ColE7 mutants in complex with the inhibitor Im7, N560A-Im7, N560D-Im7 and H573A-Im7, were determined at a resolution of 1.9 A to 2.2 A. H573 is responsible for metal ion binding in the wild-type protein, as the zinc ion is still partially associated in the structure of H573A, suggesting that H573 plays a supportive role in metal binding. Both N560A and N560D contain a disordered loop in the HNH motif due to the disruption of the hydrogen bond network surrounding the side-chain of residue 560, and as a result, the imidazole ring of the general base residue H545 is tilted slightly and the scissile phosphate is shifted, leading to the large reductions in hydrolysis activities. These results suggest that the highly conserved asparagine in the HNH motif, in general, plays a structural role in constraining the loop in the metal finger structure and keeping the general base histidine and scissile phosphate in the correct position for DNA hydrolysis.     

Histidine modulates the clastogenic effect of oxidative stress

The kinetics of the formation of single-strand breaks induced by H2O2 in DNA is more rapid in Eagle's Modified Minimal Essential Medium (MEM) than in phosphate-buffered saline (PBS). Among the components of MEM, we found that histidine increases the rate of degradation of DNA by H2O2 in PBS dose-dependently. In hamster lung fibroblasts histidine increases the cytotoxicity of H2O2, as well as the number of sister-chromatid exchanges and the frequency of micronuclei induced by hydrogen peroxide.     

Histidine 282 in 5-aminolevulinate synthase affects substrate binding and catalysis

5-Aminolevulinate synthase (ALAS), the first enzyme of the heme biosynthetic pathway in mammalian cells, is a member of the alpha-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. In all structures of the enzymes of the -oxoamine synthase family, a conserved histidine hydrogen bonds with the phenolic oxygen of the PLP cofactor and may be significant for substrate binding, PLP positioning, and maintenance of the pKa of the imine nitrogen. In ALAS, replacing the equivalent histidine, H282, with alanine reduces the catalytic efficiency for glycine 450-fold and decreases the slow phase rate for glycine binding by 85%. The distribution of the absorbing 420 and 330 nm species was altered with an A420/A330 ratio increased from 0.45 to 1.05. This shift in species distribution was mirrored in the cofactor fluorescence and 300-500 nm circular dichroic spectra and likely reflects variation in the tautomer distribution of the holoenzyme. The 300-500 nm circular dichroism spectra of ALAS and H282A diverged in the presence of either glycine or aminolevulinate, indicating that the reorientation of the PLP cofactor upon external aldimine formation is impeded in H282A. Alterations were also observed in the K(Gly)d value and spectroscopic and kinetic properties, while the K(PLP)d increased 9-fold. Altogether, the results imply that H282 coordinates the movement of the pyridine ring with the reorganization of the active site hydrogen bond network and acts as a hydrogen bond donor to the phenolic oxygen to maintain the protonated Schiff base and enhance the electron sink function of the PLP cofactor.     

HlyC, the internal protein acyltransferase that activates hemolysin toxin: role of conserved histidine, serine, and cysteine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis

HlyC is an internal protein acyltransferase that activates hemolysin, a toxic protein produced by pathogenic Escherichia coli. Acyl-acyl carrier protein (ACP) is the essential acyl donor. Separately subcloned, expressed, and purified prohemolysin A (proHlyA), HlyC, and [1-14C]myristoyl-ACP have been used to study the conversion of proHlyA to HlyA [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1998) Biochemistry 37, 4644-4655]. HlyC and hemolysin belong to a family of at least 13 toxins produced by Gram-negative bacteria. The homologous acyltransferases of the family show a number of conserved residues that are possible candidates for participation in acyl transfer. Specific chemical reagents and site-directed mutagenesis showed that neither the single conserved cysteine nor the three conserved serine residues were required for enzyme activity. Treatment with the reversible histidine-modifying diethyl pyrocarbonate (DEPC) inhibited acyltransferase activity, and acyltransferase activity was restored following hydroxylamine treatment. The substrate myristoyl-ACP protected HlyC from DEPC inhibition. These findings and spectral absorbance changes suggested that histidine, particularly a histidine proximal to the substrate binding site, was essential for enzyme activity. Site-directed mutageneses of the single conserved histidine residue, His23, to alanine, cysteine, or serine resulted in each instance in complete inactivation of the enzyme.     

JAMM: a metalloprotease-like zinc site in the proteasome and signalosome

The JAMM (JAB1/MPN/Mov34 metalloenzyme) motif in Rpn11 and Csn5 underlies isopeptidase activities intrinsic to the proteasome and signalosome, respectively. We show here that the archaebacterial protein AfJAMM possesses the key features of a zinc metalloprotease, yet with a distinct fold. The histidine and aspartic acid of the conserved EX_nHS/THX₇SXXD motif coordinate a zinc, whereas the glutamic acid hydrogen-bonds an aqua ligand. By analogy to the active site of thermolysin, we predict that the glutamic acid serves as an acid-base catalyst and the second serine stabilizes a tetrahedral intermediate. Mutagenesis of Csn5 confirms these residues are required for Nedd8 isopeptidase activity. The active site-like architecture specified by the JAMM motif motivates structure-based approaches to the study of JAMM domain proteins and the development of therapeutic proteasome and signalosome inhibitors.     

A Histidine Switch in Hemagglutinin-Neuraminidase Triggers Paramyxovirus-Cell Membrane Fusion

Most paramyxovirus fusion proteins require coexpression of and activation by a homotypic attachment protein, hemagglutinin-neuraminidase (HN), to promote membrane fusion. However, the molecular mechanism of the activation remains unknown. We previously showed that the incorporation of a monohistidylated lipid into F-virosome (Sendai viral envelope containing only fusion protein) enhanced its fusion to hepatocytes, suggesting that the histidine residue in the lipid accelerated membrane fusion. Therefore, we explored whether a histidine moiety in HN could similarly direct activation of the fusion protein. In membrane fusion assays, the histidine substitution mutants of HN (H247A of Sendai virus and H245A of human parainfluenza virus 3) had impaired membrane fusion promotion activity without significant changes in other biological activities. Synthetic 30-mer peptides corresponding to regions of the two HN proteins containing these histidine residues rescued the fusion promoting activity of the mutants, whereas peptides with histidine residues substituted by alanine did not. These histidine-containing peptides also activated F-virosome fusion with hepatocytes both in the presence and in the absence of mutant HN in the virosome. We provide evidence that the HN-mimicking peptides promote membrane fusion, revealing a specific histidine “switch” in HN that triggers fusion.     

Histidine phosphorylation in biological systems

Histidine phosphorylation is important in prokaryotes and occurs to the extent of 6% of total phosphorylation in eukaryotes. Nevertheless phosphohistidine residues are not normally observed in proteins due to rapid hydrolysis of the phosphoryl group under acidic conditions. Many rapid processes employ phosphohistidines, including the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS), the bacterial two-component systems and reactions catalyzed by enzymes such as nucleoside diphosphate kinase and succinyl-CoA synthetase. In the PTS, the NMR structure of the phosphohistidine moiety of the phosphohistidine-containing protein was determined but no X-ray structures of phosphohistidine forms of PTS proteins have been elucidated. There have been crystal structures of a few phosphohistidine-containing proteins determined: nucleoside diphosphate kinase, succinyl-CoA synthetase, a cofactor-dependent phosphoglycerate mutase and the protein PAE2307 from the hyperthermophilic archaeon Pyrobaculum aerophilum. A common theme for these stable phosphohistidines is the occurrence of ion-pair hydrogen bonds (salt bridges) involving the non-phosphorylated nitrogen atom of the histidine imidazole ring with an acidic amino acid side chain.     

Use of a Probabilistic Motif Search to Identify Histidine Phosphotransfer Domain-Containing Proteins

The wealth of newly obtained proteomic information affords researchers the possibility of searching for proteins of a given structure or function. Here we describe a general method for the detection of a protein domain of interest in any species for which a complete proteome exists. In particular, we apply this approach to identify histidine phosphotransfer (HPt) domain-containing proteins across a range of eukaryotic species. From the sequences of known HPt domains, we created an amino acid occurrence matrix which we then used to define a conserved, probabilistic motif. Examination of various organisms either known to contain (plant and fungal species) or believed to lack (mammals) HPt domains established criteria by which new HPt candidates were identified and ranked. Search results using a probabilistic motif matrix compare favorably with data to be found in several commonly used protein structure/function databases: our method identified all known HPt proteins in the Arabidopsis thaliana proteome, confirmed the absence of such motifs in mice and humans, and suggests new candidate HPts in several organisms. Moreover, probabilistic motif searching can be applied more generally, in a manner both readily customized and computationally compact, to other protein domains; this utility is demonstrated by our identification of histones in a range of eukaryotic organisms.     

Role of conserved histidine residues in metalloactivation of the ArsA ATPase

The ArsA ATPase is the catalytic subunit of a pump that is responsible for resistance to arsenicals and antimonials in Escherichia coli. Arsenite or antimonite allosterically activates the ArsA ATPase activity. ArsA homologues from eubacteria, archaea and eukarya have a signature sequence (DTAPTGHT) that includes a conserved histidine. The ArsA ATPase has two such conserved motifs, one in the NH₂-terminal (A1) half and the other in the COOH-terminal (A2) half of the protein. These sequences have been proposed to be signal transduction domains that transmit the information of metal occupancy at the allosteric to the catalytic site to activate ATP hydrolysis. The role of the conserved residues His148 and His453, which reside in the A1 and A2 signal transduction domains respectively, was investigated by mutagenesis to create H148A, H453A or H148A/H453A ArsAs. Each altered protein exhibited a decrease in the V _max of metalloid-activated ATP hydrolysis, in the order wild type ArsA>H148A>H453A>H148A/H453A. These results suggest that the histidine residues play a role in transmission of the signal between the catalytic and allosteric sites.     

The H2 sensor of Ralstonia eutropha: biochemical and spectroscopic analysis of mutant proteins modified at a conserved glutamine residue close to the [NiFe] active site

[NiFe] hydrogenases contain a highly conserved histidine residue close to the [NiFe] active site which is altered by a glutamine residue in the H(2)-sensing [NiFe] hydrogenases. In this study, we exchanged the respective glutamine residue of the H(2) sensor (RH) of Ralstonia eutropha, Q67 of the RH large subunit HoxC, by histidine, asparagine and glutamate. The replacement by histidine and asparagine resulted in slightly unstable RH proteins which were hardly affected in their regulatory and enzymatic properties. The exchange to glutamate led to a completely unstable RH protein. The purified wild-type RH and the mutant protein with the Gln/His exchange were analysed by continuous-wave and pulsed electron paramagnetic resonance (EPR) techniques. We observed a coupling of a nitrogen nucleus with the [NiFe] active site for the mutant protein which was absent in the spectrum of the wild-type RH. A combination of theoretical calculations with the experimental data provided an explanation for the observed coupling. It is shown that the coupling is due to the formation of a weak hydrogen bond between the protonated N(epsilon) nucleus of the histidine with the sulfur of a conserved cysteine residue which coordinates the metal atoms of the [NiFe] active site as a bridging ligand. The effect of this hydrogen bond on the local structure of the [NiFe] active site is discussed.     

Chemical phosphorylation of histidine-containing peptides based on the sequence of histone H4 and their dephosphorylation by protein histidine phosphatase

Using peptides based on the amino acid sequences surrounding the two histidine residues in histone H4, we have investigated the kinetics of the phosphorylation and dephosphorylation reactions of their histidine residues, when reacted with potassium phosphoramidate, by ¹H NMR. We have been able to estimate rate constants for the reactions and have shown that there are differences in the kinetics between the two peptides. The kinetics of hydrolysis of phosphoramidate was measured by ³¹P NMR and protein histidine phosphatase (PHP) was shown to catalyse the reaction. We have shown that the dephosphorylation of the phosphohistidine of the phosphopeptides is catalysed by PHP. In terms of substrate specificity, there is a small preference for 1-phosphohistidine compared to 3-phosphohistidine, although the rate accelerations for hydrolysis induced by the enzyme were 1100- and 33,333-fold, respectively. The kinetics of both the phosphorylation and dephosphorylation reactions depend on the amino acid sequence surrounding the histidine. PHP shows greater substrate specificity for the peptide whose sequence is similar to that around histidine 18 of histone H4. PHP was unable to catalyse the dephosphorylation of histone H4 that had been phosphorylated with a histone H4 histidine kinase.     

Essential role of histidine 84 in elongation factor Tu for the chemical step of GTP hydrolysis on the ribosome

Elongation factor Tu (EF-Tu) is a GTP-binding protein that delivers aminoacyl-tRNA to the A site of the ribosome during protein synthesis. The mechanism of GTP hydrolysis in EF-Tu on the ribosome is poorly understood. It is known that mutations of a conserved histidine residue in the switch II region of the factor, His84 in Escherichia coli EF-Tu, impair GTP hydrolysis. However, the partial reaction which is directly affected by mutations of His84 was not identified and the effect on GTP hydrolysis was not quantified. Here, we show that the replacement of His84 with Ala reduces the rate constant of GTP hydrolysis more than 10(6)-fold, whereas the preceding steps of ternary complex binding to the ribosome, codon recognition and, most importantly, the GTPase activation step are affected only slightly. These results show that His84 plays a key role in the chemical step of GTP hydrolysis. Rate constants of GTP hydrolysis by wild-type EF-Tu, measured using the slowly hydrolyzable GTP analog, GTPgammaS, showed no dependence on pH, indicating that His84 does not act as a general base. We propose that the catalytic role of His84 is to stabilize the transition state of GTP hydrolysis by hydrogen bonding to the attacking water molecule or, possibly, the gamma-phosphate group of GTP.     

Architecture of the catalytic HPN motif is conserved in all E2 conjugating enzymes

E2 conjugating enzymes are the central enzymes in the ubiquitination pathway and are responsible for the transfer of ubiquitin and ubiquitin-like proteins on to target substrates. The secondary structural elements of the catalytic domain of these enzymes is highly conserved, including the sequence conservation of a three-residue HPN (His-Pro-Asn) motif located upstream of the active-site cysteine residue used for ubiquitin conjugation. Despite the vast structural knowledge of E2 enzymes, the catalytic mechanism of these enzymes remains poorly understood, in large part due to variation in the arrangements of the residues in the HPN motif in existing E2 structures. In the present study, we used the E2 enzyme HIP2 to probe the structures of the HPN motif in several other E2 enzymes. A combination of chemical-shift analysis, determination of the histidine protonation states and amide temperature coefficients were used to determine the orientation of the histidine ring and hydrogen-bonding arrangements within the HPN motif. Unlike many three-dimensional structures, we found that a conserved hydrogen bond between the histidine imidazole ring and the asparagine backbone amide proton, a common histidine protonation state, and a common histidine orientation exists for all E2 enzymes examined. These results indicate that the histidine within the HPN motif is orientated to structurally stabilize a tight turn motif in all E2 enzymes and is not orientated to interact with the asparagine side chain as proposed in some mechanisms. These results suggest that a common catalysis mechanism probably exists for all E2 conjugating enzymes to facilitate ubiquitin transfer.     

Topological Orientation of Acyl-CoA:Diacylglycerol Acyltransferase-1 (DGAT1) and Identification of a Putative Active Site Histidine and the Role of the N Terminus in Dimer/Tetramer Formation

Acyl CoA:diacylglycerol acyltransferase (DGAT) is an integral membrane protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerols. Two DGAT enzymes have been identified (DGAT1 and DGAT2) with unique roles in lipid metabolism. DGAT1 is a multifunctional acyltransferase capable of synthesizing diacylglycerol, retinyl, and wax esters in addition to triacylglycerol. Here, we report the membrane topology for murine DGAT1 using protease protections assays and indirect immunofluorescence in conjunction with selective permeabilization of cellular membranes. Topology models based on prediction algorithms suggested that DGAT1 had eight transmembrane domains. In contrast, our data indicate that DGAT1 has three transmembrane domains with the N terminus oriented toward the cytosol. The C-terminal region of DGAT1, which accounts for ∼50% of the protein, is present in the endoplasmic reticulum lumen and contains a highly conserved histidine residue (His-426) that may be part of the active site. Mutagenesis of His-426 to alanine impaired the ability of DGAT1 to synthesize triacylglycerols as well as retinyl and wax esters in an in vitro acyltransferase assay. Finally, we show that the N-terminal domain of DGAT1 is not required for the catalytic activity of DGAT1 but, instead, may be involved in regulating enzyme activity and dimer/tetramer formation.     

Solution structure of the low-molecular-weight protein tyrosine phosphatase from Tritrichomonas foetus reveals a flexible phosphate binding loop

Eukaryotic low-molecular-weight protein tyrosine phosphatases (LMW PTPs) contain a conserved serine, a histidine with an elevated pKa, and an active site asparagine that together form a highly conserved hydrogen bonding network. This network stabilizes the active site phosphate binding loop for optimal substrate binding and catalysis. In the phosphatase from the bovine parasite Tritrichomonas foetus (TPTP), both the conserved serine (S37) and asparagine (N14) are present, but the conserved histidine has been replaced by a glutamine residue (Q67). Site-directed mutagenesis, kinetic, and spectroscopic experiments suggest that Q67 is located near the active site and is important for optimal catalytic activity. Kinetic experiments also suggest that S37 participates in the active site/hydrogen bonding network. Nuclear magnetic resonance spectroscopy was used to determine the three-dimensional structure of the TPTP enzyme and to further examine the roles of S37 and Q67. The backbone conformation of the TPTP phosphate binding loop is nearly superimposable with that of other tyrosine phosphatases, with N14 existing in a strained, left-handed conformation that is a hallmark of the active site hydrogen bonding network in the LMW PTPs. As expected, both S37 and Q67 are located at the active site, but in the consensus structure they are not within hydrogen bonding distance of N14. The hydrogen bond interactions that are observed in X-ray structures of LMW PTPs may in fact be transient in solution. Protein dynamics within the active site hydrogen bonding network appear to be affected by the presence of substrate or bound inhibitors such as inorganic phosphate.     

Stereochemical retention of the configuration in the action of Fhit on phosphorus-chiral substrates

Fhit is the protein product of FHIT, a candidate human tumor suppressor gene. Fhit catalyzes the hydrolysis of diadenosine triphosphate (Ap3A) to AMP and ADP. Fhit is here shown to catalyze the hydrolysis in H218O with production of adenosine 5'-[18O]phosphate and ADP, proving that the substitution of water is at Palpha and not at Pbeta. The chain fold of Fhit is similar to that of galactose-1-phosphate uridylyltransferase, which functions by a double-displacement mechanism through the formation of a covalent nucleotidyl-enzyme intermediate and overall retention of configuration at Palpha. The active site of Fhit contains a histidine motif that is reminiscent of the HPH motif in galactose-1-phosphate uridylyltransferases, in which the first histidine residue serves as the nucleophilic catalyst to which the nucleotidyl group is bonded covalently in the covalent intermediate. In this work, the Fhit-catalyzed cleavage of (RP)- and (SP)-gamma-(m-nitrobenzyl) adenosine 5'-O-1-thiotriphosphate (mNBATPalphaS) in H218O to adenosine 5'-[18O]thiophosphate is shown to proceed with overall retention of configuration at phosphorus. gamma-(m-Nitrobenzyl) adenosine 5'-O-triphosphate (mNBATP) is approximately as good a substrate for Fhit as Ap3A, and both (RP)- and (SP)-mNBATPalphaS are substrates that react at about 0.5% of the rate of Ap3A. The stereochemical evidence indicates that hydrolysis by Fhit proceeds by a double-displacement mechanism, presumably through a covalent AMP-enzyme intermediate.     

Structure-function analysis of a novel member of the LIV-1 subfamily of zinc transporters, ZIP14

Here, we report the first investigation of a novel member of the LZT (LIV-1 subfamily of ZIP zinc Transporters) subfamily of zinc influx transporters. LZT subfamily sequences all contain a unique and highly conserved metalloprotease motif (HEXPHEXGD) in transmembrane domain V with both histidine residues essential for zinc transport by ZIP (Zrt-, Irt-like Proteins) transporters. We investigate here whether ZIP14 (SLC39A14), lacking the initial histidine in this motif, is still able to transport zinc. We demonstrate that this plasma membrane located glycosylated protein functions as a zinc influx transporter in a temperature-dependant manner.     

Integron integrases possess a unique additional domain necessary for activity.

Integrons are genetic elements capable of integrating genes by a site-specific recombination system catalyzed by an integrase. Integron integrases are members of the tyrosine recombinase family and possess the four invariant residues (RHRY) and conserved motifs (boxes I and II and patches I, II, and III). An alignment of integron integrases compared to other tyrosine recombinases shows an additional group of residues around the patch III motif. We have analyzed the DNA binding and recombination properties of class I integron integrase (IntI1) variants carrying mutations at residues that are well conserved among all tyrosine recombinases and at some residues from the additional motif that are conserved among the integron integrases. The well-conserved residues studied were H277 from the conserved tetrad RHRY (about 90% conserved), E121 found in the patch I motif (about 80% conserved in prokaryotic recombinases), K171 from the patch II motif (near 100% conserved), W229 and F233 from the patch III motif, and G302 of box II (about 80% conserved in prokaryotic recombinases). Additional IntI1 mutated residues were K219 and a deletion of the sequence ALER215. We observed that E121, K171, and G302 play a role in the recombination activity but can be mutated without disturbing binding to DNA. W229, F233, and the conserved histidine (H277) may be implicated in protein folding or DNA binding. Some of the extra residues of IntI1 seem to play a role in DNA binding (K219) while others are implicated in the recombination activity (ALER215 deletion).