Two histidine residues are essential for catalysis by lecithin retinol acyl transferase

Lecithin retinol acyl transferase (LRAT) is a novel membrane bound enzyme that catalyzes the formation of retinyl esters from vitamin A and lecithin. The enzyme is both essential for vision and for the general mobilization of vitamin A. The sequence of LRAT defines it as a novel enzyme unrelated to any other protein of known function. LRAT possesses a catalytically essential active site cysteine residue. The enzyme also contains six histidine residues. It is shown here that two of these residues (H57 and H163) are essential for catalysis. A mechanistic hypothesis is presented to account for these observations.     

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.     

Structurally conserved interaction of Lgl family with SNAREs is critical to their cellular function

The Lethal giant larvae (Lgl) tumor suppressor family is conserved from yeast to mammals and plays a critical yet controversial role in cell polarity. Studies on Drosophila Lgl suggest that its function in polarity is through regulation of the acto-myosin cytoskeleton. In contrast, studies on the yeast Lgl homologs, Sro7/Sro77, suggest a function in exocytosis through interaction with the t-SNARE Sec9. Using yeast/mammalian Lgl chimeras, we demonstrate that the overall architecture of Lgl proteins is highly conserved and that the C-terminal domain is the major site of SNARE interaction within both yeast and mammalian homologs. Importantly, we find that the ability of Lgl chimeras to function as the only source of Lgl in yeast correlates precisely with the ability to interact with the yeast t-SNARE. We report a novel interaction between Sro7 and the yeast myosin V, Myo2. However, we find that interactions with either Myo2 or Myo1 (myosin II) cannot account for the dramatic functional differences observed for these chimeras in yeast. These results provide the first demonstration that the interaction of an Lgl family member with a specific effector is critical to its function in vivo. These data support the model that the Lgl family functions in cell polarity, at least in part, by regulating SNARE-mediated membrane delivery events at the cell surface.     

Two highly conserved glutamate residues critical for type III sodium-dependent phosphate transport revealed by uncoupling transport function from retroviral receptor function

Type III sodium-dependent phosphate (NaP(i)) cotransporters, Pit1 and Pit2, have been assigned housekeeping P(i) transport functions and suggested involved in chondroblastic and osteoblastic mineralization and ectopic calcification. Both proteins exhibit dual function, thus, besides being transporters, they also serve as receptors for several gammaretroviruses. We here show that it is possible to uncouple transport and receptor functions of a type III NaP(i) cotransporter and thus exploit the retroviral receptor function as a control for proper processing and folding of mutant proteins. Thus exchanging two putative transmembranic glutamate residues in human Pit2, Glu(55) and Glu(575), with glutamine or with lysine severely impaired or knocked out, respectively, P(i) transport function, but left viral receptor function undisturbed. Both glutamates are conserved in type III NaP(i) cotransporters, in fungal NaP(i) cotransporters PHO-4 and Pho89, and in other known or putative phosphate permeases from a number of species and are the first residues shown to be critical for type III NaP(i) cotransport. Their putative transmembranic positions together with the presented data are consistent with Glu(55) and Glu(575) being parts of a cation liganding site or playing roles in conformational changes associated with substrate transport. Finally, the results also show that Pit2 retroviral receptor function per se is not dependent on Pit2 P(i) transport function.     

Mutagenesis of histidinol dehydrogenase reveals roles for conserved histidine residues.

The dimeric zinc metalloenzyme L-histidinol dehydrogenase (HDH) catalyzes an unusual four-electron oxidation of the amino alcohol histidinol via the histidinaldehyde intermediate to the acid product histidine with the reduction of two molecules of NAD. An essential base, with pKa about 8, is involved in catalysis. Here we report site-directed mutagenesis studies to replace each of the five histidine residues (His-98, His-261, His-326, His-366, and His-418) in Salmonella typhimurium with either asparagine or glutamine. In all cases, the overexpressed enzymes were readily purified and behaved as dimers. Substitution of His-261 and His-326 by asparagine caused about 7000- and 500-fold decreases in kcat, respectively, with little change in KM values. Similar loss of activity was also reported for a H261N mutant Brassica HDH [Nagai, A., and Ohta, D. (1994) J. Biochem. 115, 22-25]. Kinetic isotope effects, pH profiles, substrate rescue, and stopped-flow experiments suggested that His-261 and His-326 are involved in proton transfers during catalysis. Sensitivity to metal ion chelator and decreased affinities for metal ions with substitutions at His-261 and His-418 suggested that these two residues are candidates for zinc ion ligands.     

Two types of transmembrane homomeric interactions in the integrin receptor family are evolutionarily conserved

Integrins are heterodimers, but recent in vitro and in vivo experiments suggest that they are also able to associate through their transmembrane domains to form homomeric interactions. Two fundamental questions are the biological relevance of these aggregates and their form of interaction in the membrane domain. Although in vitro experiments have shown the involvement of a GxxxG-like motif, several crosslinking in vivo data are consistent with an almost opposite form of interaction between the transmembrane alpha-helices. In the present work, we have explored these two questions using molecular dynamics simulations for all available integrin types. We have tested the hypothesis that homomeric interactions are evolutionary conserved, and essential for the cell, using conservative substitutions to filter out nonnative interactions. Our results show that two models, one involving a GxxxG-like motif (model I) and an almost opposite form of interaction (model II) are conserved across all alpha and beta integrin types, both in homodimers and homotrimers, with different specificities. No conserved interaction was found for homotetramers. Our results are completely independent from experimental data, both during molecular dynamics simulations and in the selection of the correct models. We rationalize previous seemingly conflicting findings regarding the nature of integrin interhelical homomeric interactions.     

Conserved serine and histidine residues are critical for activity of the ER-type signal peptidase SipW of Bacillus subtilis

Type I signal peptidases (SPases) are required for the removal of signal peptides from translocated proteins and, subsequently, release of the mature protein from the trans side of the membrane. Interestingly, prokaryotic (P-type) and endoplasmic reticular (ER-type) SPases are functionally equivalent, but structurally quite different, forming two distinct SPase families that share only few conserved residues. P-type SPases were, so far, exclusively identified in eubacteria and organelles, whereas ER-type SPases were found in the three kingdoms of life. Strikingly, the presence of ER-type SPases appears to be limited to sporulating Gram-positive eubacteria. The present studies were aimed at the identification of potential active site residues of the ER-type SPase SipW of Bacillus subtilis, which is required for processing of the spore-associated protein TasA. Conserved serine, histidine, and aspartic acid residues are critical for SipW activity, suggesting that the ER-type SPases employ a Ser-His-Asp catalytic triad or, alternatively, a Ser-His catalytic dyad. In contrast, the P-type SPases employ a Ser-Lys catalytic dyad (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186-190). Notably, catalytic activity of SipW was not only essential for pre-TasA processing, but also for the incorporation of mature TasA into spores.     

Two histidine residues are essential for ribonuclease T1 activity as is the case for ribonuclease A

Ribonuclease T1 (RNase T1, EC 3.1.27.3) is a guanosine-specific ribonuclease that cleaves the 3',5'-phosphodiester linkage of single-stranded RNA. It is assumed that the reaction is generated by concerted acid-base catalysis between residues Glu-58 and His-92 or His-40. From the results of chemical modification and NMR studies, it appeared that the residue Glu-58 was indispensable for nucleolytic activity. However, we have recently demonstrated that Glu-58 is an important but not an essential residue for catalytic activity, using the methods of genetic engineering to change Glu-58 to Gln-58 etc [Nishikawa, S., Morioka, H., Fuchimura, K., Tanaka, T., Uesugi, S., Ohtsuka, E., & Ikehara, M. (1986) Biochem. Biophys. Res. Commun. 138, 789-794]. In the present paper, we report that mutants of RNase T1 with residue Ala-40 or Ala-92 have almost no activity, while mutants that contain Ala-58 retain considerable activity. These results show that the two histidine residues, His-40 and His-92, but not Glu-58, are indispensable for the catalytic activity of the enzyme. We propose a revised reaction mechanism in which two histidine residues play a major role, as they do in the case of RNase A.     

Members of a unique histidine acid phosphatase family are conserved amongst a group of primitive eukaryotic human pathogens

Recently, we identified and characterized the genes encoding several distinct members of the histidine-acid phosphatase enzyme family from Leishmania donovani, a primitive protozoan pathogen of humans. These included genes encoding the heavily phosphorylated/glycosylated, tartrate-sensitive, secretory acid phosphatases (Ld SAcP-1 and Ld SAcP-2) and the unique, tartrate-resistant, externally-oriented, surface membrane-bound acid phosphatase (Ld MAcP) of this parasite. It had been previously suggested that these enzymes may play essential roles in the growth, development and survival of this organism. In this report, to further examine this hypothesis, we assessed whether members of the L. donovani histidine-acid phosphatase enzyme family were conserved amongst other pathogenic Leishmania and related trypanosomatid parasites. Such phylogenetic conservation would clearly indicate an evolutionary selection for this family of enzymes and strongly suggest and support an important functional role for acid phosphatases to the survival of these parasites. Results of pulsed field gel electrophoresis and Southern blotting showed that homologs of both the Ld SAcPs and Ld MAcP were present in each of the visceral and cutaneous Leishmania species examined (i.e. isolates of L. donovani, L. infantum,L. tropica, L. major and L. mexicana, respectively). Further, results of enzyme assays showed that all of these organisms expressed both tartrate-sensitive and tartrate-resistant acid phosphatase activities. In addition, homologs of both the Ld SAcPs and Ld MAcP genes and their corresponding enzyme activities were also identified in two Crithidia species (C. fasciculata and C. luciliae) and in Leptomonas seymouri. In contrast, Trypanosoma brucei, Trypanosoma cruzi and Phytomonas serpens had only very low levels of such enzyme activities. Cumulatively, results of this study showed that homologs of the Ld SAcPs and Ld MAcP are conserved amongst all pathogenic Leishmania sps. suggesting that they may play significant functional roles in the growth, development and survival of all members of this important group of human pathogens.     

A conserved histidine in vertebrate-type ferredoxins is critical for redox-dependent dynamics

Adrenodoxin (Adx) belongs to the family of Cys(4)Fe(2)S(2) vertebrate-type ferredoxins that shuttle electrons from NAD(P)H-dependent reductases to cytochrome P450 enzymes. The vertebrate-type ferredoxins contain a conserved basic residue, usually a histidine, adjacent to the third cysteine ligand of the Cys(4)Fe(2)S(2) cluster. In bovine Adx the side chain of this residue, His 56, is involved in a hydrogen-bonding network within the domain of Adx that interacts with redox partners. It has been proposed that this network acts as a mechanical link between the metal cluster binding site and the interaction domain, transmitting redox-dependent conformational or dynamical changes from the cluster binding loop to the interaction domain. H/D exchange studies indicate that oxidized Adx (Adx(o)) is more dynamic than reduced Adx (Adx(r)) on the kilosecond time scale in many regions of the protein, including the interaction domain. Dynamical differences on picosecond to nanosecond time scales between the oxidized (Adx(o)) and reduced (Adx(r)) adrenodoxin were probed by measurement of (15)N relaxation parameters. Significant differences between (15)N R(2) rates were observed for all residues that could be measured, with those rates being faster in Adx(o) than in Adx(r). Two mutations of His 56, H56R and H56Q, were also characterized. No systematic redox-dependent differences between (15)N R(2) rates or H/D exchange rates were observed in either mutant, indicating that His 56 is required for the redox-dependent behavior observed in WT Adx. Comparison of chemical shift differences between oxidized and reduced H56Q and H56R Adx confirms that redox-dependent changes are smaller in these mutants than in the wild-type Adx.     

Role of the four conserved histidine residues in the amidotransferase domain of carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from ATP, bicarbonate, and glutamine. The amidotransferase activity of this enzyme is catalyzed by the smaller of the two subunits of the heterodimeric protein. The roles of four conserved histidine residues within this subunit were probed by site-directed mutagenesis to asparagine. The catalytic activities of the H272N and H341N mutants are not significantly different than that of the wild-type enzyme. The H353N mutant is unable to utilize glutamine as a nitrogen source in the synthetase reaction or the partial glutaminase reaction. However, binding to the glutamine active site is not impaired in the H353N enzyme since glutamine is found to activate the partial ATPase reaction by 40% with a Kd of 54 microM. The H312N mutant has a Michaelis constant for glutamine that is 2 orders of magnitude larger than the wild-type value, but the maximal rate of glutamine hydrolysis is unchanged. These results are consistent with His-353 functioning as a general acid/base catalyst for proton transfers while His-312 serves a critical role for the binding of glutamine to the active site.     

Two conserved tryptophan residues of tumor necrosis factor and lymphotoxin are not involved in the biological activity

Each of the two highly conserved tryptophan residues in hTNF (positions 28 and 114) was converted into phenylalanine by site-directed mutagenesis and the mutant proteins were partially purified. A cytotoxicity assay on mouse L929 cells showed only a slight reduction in biological activity, strongly suggesting that neither of the two amino acids is involved in the active site.     

Role of histidine residues in the binding mechanism of the growth hormone family

1. Reactivity of the hGH histidine residues were studied by reaction with ethoxyformic anhydride. Localization in the molecule of three kinetically distinguishable classes, each including only one residue, was achieved. 2. The first was composed of residue 151, with an apparent velocity constant k = 0.735/min, (similar to that of histidines 19 and 21 in bGH and eGH). The second histidine, 18, with a velocity constant k = 0.135/min, (similar to that of histidine 169 in the above hormones), and a third, histidine 21, which does not react at all. 3. Neither histidine 151 nor 18 seem to be involved, at least not directly, in bGH binding to specific rat liver sites, since the decrease in this capacity was only 47% after modification of the former by 77 and 65% after total modification of the latter. 4. These results, and those previously obtained with bGH and eGH, suggest that either histidine 21 is the only indispensable histidine for the binding of growth hormones to specific rat liver sites, or that histidine 21 and/or 18 (19 in bGH and eGH), are located within the growth hormone binding site interaction area.     

Effect of mutations of five conserved histidine residues in the catalytic subunit of the cbb3 cytochrome c oxidase on its function

The cbb3 cytochrome c oxidase has the dual function as a terminal oxidase and oxygen sensor in the photosynthetic bacterium, Rhodobacter sphaeroides. The cbb3 oxidase forms a signal transduction pathway together with the PrrBA two-component system that controls photosynthesis gene expression in response to changes in oxygen tension in the environment. Under aerobic conditions the cbb3 oxidase generates an inhibitory signal, which shifts the equilibrium of PrrB kinase/phosphatase activities towards the phosphatase mode. Photosynthesis genes are thereby turned off under aerobic conditions. The catalytic subunit (CcoN) of the R. sphaeroides cbb3 oxidase contains five histidine residues (H214, H233, H303, H320, and H444) that are conserved in all CcoN subunits of the cbb3 oxidase, but not in the catalytic subunits of other members of copper-heme superfamily oxidases. H214A mutation of CcoN affected neither catalytic activity nor sensory (signaling) function of the cbb3 oxidase, whereas H320A mutation led to almost complete loss of both catalytic activity and sensory function of the cbb3 oxidase. H233V and H444A mutations brought about the partial loss of catalytic activity and sensory function of the cbb3 oxidase. Interestingly, the H303A mutant form of the cbb3 oxidase retains the catalytic function as a cytochrome c oxidase as compared to the wild-type oxidase, while it is defective in signaling function as an oxygen sensor. H303 appears to be implicated in either signal sensing or generation of the inhibitory signal to the PrrBA two-component system.     

Quorum sensing in Staphylococci is regulated via phosphorylation of three conserved histidine residues

Staphylococcus aureus cause infections by producing toxins, a process regulated by cell-cell communication (quorum sensing) through the histidine-phosphorylation of the target of RNAIII-activating protein (TRAP). We show here that TRAP is highly conserved in staphylococci and contains three completely conserved histidine residues (His-66, His-79, His-154) that are phosphorylated and essential for its activity. This was tested by constructing a TRAP(-) strain with each of the conserved histidine residues changed to alanine by site-directed mutagenesis. All mutants were tested for pathogenesis in vitro (expression of RNAIII and hemolytic activity) and in vivo (murine cellulitis model). Results show that RNAIII is not expressed in the TRAP(-) strain, that it is non hemolytic, and that it does not cause disease in vivo. These pathogenic phenotypes could be rescued in the strain containing the recovered traP, confirming the importance of TRAP in S. aureus pathogenesis. The phosphorylation of TRAP mutated in any of the conserved histidine residues was significantly reduced, and mutants defective in any one of these residues were non-pathogenic in vitro or in vivo, whereas those mutated in a non-conserved histidine residue (His-124) were as pathogenic as the wild type. These results confirm the importance of the three conserved histidine residues in TRAP activity. The phosphorylation pattern, structure, and gene organization of TRAP deviates from signaling molecules known to date, suggesting that TRAP belongs to a novel class of signal transducers.     

Clarifying the catalytic roles of conserved residues in the amidase signature family

Fatty acid amide hydrolase (FAAH) is a mammalian integral membrane enzyme responsible for the hydrolysis of a number of neuromodulatory fatty acid amides, including the endogenous cannabinoid anandamide and the sleep-inducing lipid oleamide. FAAH belongs to a large class of hydrolytic enzymes termed the "amidase signature family," whose members are defined by a conserved stretch of approximately 130 amino acids termed the "amidase signature sequence." Recently, site-directed mutagenesis studies of FAAH have targeted a limited number of conserved residues in the amidase signature sequence of the enzyme, identifying Ser-241 as the catalytic nucleophile and Lys-142 as an acid/base catalyst. The roles of several other conserved residues with potentially important and/or overlapping catalytic functions have not yet been examined. In this study, we have mutated all potentially catalytic residues in FAAH that are conserved among members of the amidase signature family, and have assessed their individual roles in catalysis through chemical labeling and kinetic methods. Several of these residues appear to serve primarily structural roles, as their mutation produced FAAH variants with considerable catalytic activity but reduced expression in prokaryotic and/or eukaryotic systems. In contrast, five mutations, K142A, S217A, S218A, S241A, and R243A, decreased the amidase activity of FAAH greater than 100-fold without detectably impacting the structural integrity of the enzyme. The pH rate profiles, amide/ester selectivities, and fluorophosphonate reactivities of these mutants revealed distinct catalytic roles for each residue. Of particular interest, one mutant, R243A, displayed uncompromised esterase activity but severely reduced amidase activity, indicating that the amidase and esterase efficiencies of FAAH can be functionally uncoupled. Collectively, these studies provide evidence that amidase signature enzymes represent a large class of serine-lysine catalytic dyad hydrolases whose evolutionary distribution rivals that of the catalytic triad superfamily.     

Two conserved residues are important for inducing highly ordered membrane domains by the transmembrane domain of influenza hemagglutinin

The interaction with lipids of a synthetic peptide corresponding to the transmembrane domain of influenza hemagglutinin was investigated by means of electron spin resonance. A detailed analysis of the electron spin resonance spectra from spin-labeled phospholipids revealed that the major effect of the peptide on the dynamic membrane structure is to induce highly ordered membrane domains that are associated with electrostatic interactions between the peptide and negatively charged lipids. Two highly conserved residues in the peptide were identified as being important for the membrane ordering effect. Aggregation of large unilamellar vesicles induced by the peptide was also found to be correlated with the membrane ordering effect of the peptide, indicating that an increase in membrane ordering, i.e., membrane dehydration, is important for vesicle aggregation. The possibility that hydrophobic interaction between the highly ordered membrane domains plays a role in vesicle aggregation and viral fusion is discussed.     

Two conserved tryptophan residues are responsible for intrinsic fluorescence enhancement in Rubisco activase upon ATP binding

Two species-invariant tryptophan residues at positions 109 and 250 of tobacco Rubisco activase were identified by site-directed mutagenesis as being responsible for the increase in intrinsic fluorescence upon addition of ATP, which has been previously attributed to increased self-association. Substitution of W109, which is immediately prior to a ‘P-loop’ sequence in the ATP catalytic motif, with aromatic residues (Tyr or Phe), Cys or Lys eliminated both ATP hydrolysis and the intrinsic fluorescence enhancement. Although the W109 mutants bound ATP, ATP did not provide a partial protection against proteolysis by trypsin that was observed with the recombinant wild-type enzyme. In contrast, substitution of W250 with Tyr or Phe abolished about half (44%) of the increase in intrinsic fluorescence with ATP, but had little effect on ATP hydrolysis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation or proteolytic protection with ATP. The substitution of the other tryptophan residues, W16 and W305, with phenylalanine did not significantly alter the change in intrinsic fluorescence upon addition of ATP. Therefore, W109 and W250 are the residues reporting the conformational change that increases the intrinsic fluorescence.     

The role of histidine residues conserved in the putative ATP-binding region of macrolide 2'-phosphotransferase II

Macrolide 2'-phosphotransferase (MPH(2')) catalyzes the transfer of the gamma-phosphate of ATP to the 2'-hydroxyl group of macrolide antibiotics. In this study, H198 and H205, conserved in the ATP-binding region motif 1 in the putative amino acid sequence of MPH(2')II, were replaced by Ala to investigate their role. H205 was also subsequently replaced by Asn. H198A and H205N mutant enzymes retained more than 50% of the specific activity of the original enzyme to substrate oleandomycin. On the other hand, the specific activity of the H205A mutant enzyme was reduced to less than 1% of that of the wild enzyme. The results suggested that H205 is crucial for maintaining the catalytic activity of MPH(2')II, and Asn can substitute for His at this position.     

Polar residues in a conserved motif spanning helices 1 and 2 are functionally important in the SulP transporter family

The SulP family (including the SLC26 family) is a diverse family of anion transporters found in all domains of life, with different members transporting different anions. We used sequence and bioinformatics analysis of helices 1 and 2 of SulP family members to identify a conserved motif, extending the previously defined 'sulfate transporter motif'. The analysis showed that in addition to being highly conserved in both sequence and spacing, helices 1 and 2 contain a significant number of polar residues and are predicted to be buried within the protein interior, with at least some faces packed closely against other helices. This suggests a significant functional role for this region and we tested this by mutating polar residues in helices 1 and 2 in the sulfate transporter, SHST1. All mutations made, even those removing only a single hydroxyl group, had significant effects on transport. Many mutations abolished transport without affecting plasma membrane expression of the mutant protein, suggesting a functional role for these residues. Different helical faces appear to have different roles, with the most severe effects being localised to two interacting faces of helices 1 and 2. Our results confirm the predicted importance of conserved polar residues in helices 1 and 2 and suggest that transport of sulfate by SHST1 is dependent on a network of polar and aromatic interactions between these two helices.