Biosynthetic control of molecular weight in the polymerization of the octasaccharide subunits of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti

Succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti, is composed of polymerized octasaccharide subunits, each of which consists of one galactose and seven glucoses with succinyl, acetyl, and pyruvyl modifications. Production of specific low molecular weight forms of R. meliloti exported and surface polysaccharides, including succinoglycan, appears to be important for nodule invasion. In a previous study of the roles of the various exo gene products in succinoglycan biosynthesis, exoP, exoQ, and exoT mutants were found to synthesize undecaprenol-linked fully modified succinoglycan octasaccharide subunits, suggesting possible roles for their gene products in polymerization or transport. Using improved techniques for analyzing succinoglycan biosynthesis by these mutants, we have obtained evidence indicating that R. meliloti has genetically separable systems for the synthesis of high molecular weight succinoglycan and the synthesis of a specific class of low molecular weight oligosaccharides consisting of dimers and trimers of the octasaccharide subunit. Models to account for our unexpected findings are discussed. Possible roles for the ExoP, ExoQ, and ExoT proteins are compared and contrasted with roles that have been suggested on the basis of homologies to key proteins involved in the biosynthesis of O-antigens and of certain exported or capsular cell surface polysaccharides.     

Stability studies of amino acid substitutions at tyrosine 27 of the staphylococcal nuclease beta-barrel

In order to help determine the extent to which side chain interactions within the staphylococcal nuclease beta-barrel affect its global stability, a full set of point mutants was generated for residue 27. Intrinsic tryptophan fluorescence was monitored during solvent denaturation with guanidine hydrochloride (GuHCl) and was used to calculate DeltaGH2O unfolding and m values for each mutant. In the wild type protein, residue 27 is a tyrosine which is at the first position of a type I' beta-turn, and which participates in both hydrophobic interactions and side chain to side chain hydrogen bonding. The hydrophobicity of the mutant residue was found to be the dominant factor in determining global protein stability within this series of nuclease mutants.     

Specific substitutions at amino acid 256 of the sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPase mediate resistance to thapsigargin in thapsigargin-resistant hamster cells

High levels of resistance to thapsigargin (TG), a specific inhibitor of intracellular Ca2+ transport ATPases (SERCAs), can be developed in culture by stepwise exposure of mammalian cells to increasing concentrations of TG. We have identified, in two independently selected TG-resistant hamster cell lines of different lineages, mutant forms of SERCA. In the TG-resistant Chinese hamster lung fibroblast cell line DC-3F/TG, a T --> C change at nucleotide 766 introduces a Phe256 --> Leu alteration within the first cytosolic loop of the SERCA. In contrast, in the TG-resistant Syrian hamster smooth muscle cell line DDT/TG 4 microM, a T --> C change at nucleotide 767 introduces a Phe256 --> Ser mutation at that position. When these specific mutations are introduced into a wild-type full-length avian SERCA1 cDNA, transfection experiments reveal that Ca2+ transport function and ATP hydrolytic activity are not altered by such mutations. However, a 4-5-fold resistance to TG inhibition of Ca2+ transport function occurs upon the introduction of either the Phe256 --> Leu or the Phe256 --> Ser mutation into wild-type SERCA1. These specific mutations also render the hydrolytic activity of the ATPase resistant to inhibition by TG. Our results not only implicate amino acid 256 in TG-SERCA interactions, but also demonstrate that specific mutations within SERCA can mediate resistance to TG.     

Single amino acid substitutions disrupt tetramer formation in the dihydroneopterin aldolase enzyme of Pneumocystis carinii

In the opportunistic pathogen Pneumocystis carinii, dihydroneopterin aldolase function is expressed as the N-terminal portion of the multifunctional folic acid synthesis protein (Fas). This region encompasses two domains, FasA and FasB, which are 27% amino acid identical. FasA and FasB also share significant amino acid sequence similarity with bacterial dihydroneopterin aldolases. In the present study, this enzyme function has been overproduced as an independent monofunctional activity in Escherichia coli. Recombinant FasAB-Met23 (amino acids 23-290 of the predicted open reading frame) was purified and shown to contain dihydroneopterin aldolase activity. The native FasAB-Met23 is a tetramer of the 30-kDa subunit, demonstrating characteristics of an associating-dissociating equilibrium system in which only the multimeric form of the enzyme is active. Multiple sequence alignment of FasA and FasB with other dihydroneopterin aldolases highlights only three positions where the amino acid is invariable between all the predicted proteins. The role of these conserved amino acid residues in enzyme function was investigated using site-directed mutagenesis. Mutant FasAB-Met23 species were overproduced and purified to near homogeneity. Three FasA domain mutants and two FasB domain mutants had little or no detectable dihydroneopterin aldolase activity, implicating both FasA and FasB in the catalytic mechanism. We show that each mutant protein containing an inactivating amino acid substitution has lost its ability to form stable tetramers.     

Thyrotropin receptor cleavage at site 1 does not involve a specific amino acid motif but instead depends on the presence of the unique, 50 amino acid insertion

Thyrotropin (TSH) receptor (TSHR) A and B subunits are formed by intramolecular cleavage of the single chain receptor at two separate sites. The region involved in cleavage at Site 2 has been identified, but previous mutagenesis studies failed to identify Site 1. We now report fortuitous observations on the effect of trypsin on the TSHR that localizes a small region harboring Site 1. Thus, as detected by immunoblotting and by 125I-TSH cross-linking to TSHR expressed on the surface of intact CHO cells, trypsin clipped a small polypeptide fragment bearing a glycan moiety from the C terminus of the A subunit. Based on the TSHR primary structure, this small fragment (1-2 kDa) contains Asn-302. This information, together with estimation of the size of the deglycosylated A subunit relative to a series of C-terminal truncated TSHR ectodomain variants, places cleavage Site 1 in the vicinity of, or closely upstream to, residue 317. Remarkably, mutagenesis of every amino acid residue between residues 298-316 (present study) and 317-362 (previous data) did not prevent cleavage at Site 1. However, cleavage at this site was abrogated by deletion of a 50-amino acid segment (residues 317-366) unique to the TSHR in the glycoprotein hormone receptor family. In summary, these data provide novel insight into TSHR intramolecular cleavage. Cleavage at Site 1 does not depend on a specific amino acid motif and differs from cleavage at Site 2 by involvement of a mechanism requiring the presence of the enigmatic TSHR 50-amino acid "insertion."     

Analysis of the site occupancy constraints of primary amino acid sequences in the motif directing palmitylation of the vaccinia virus 37-kDa envelope protein

Vaccinia virus (VV) encodes a 37-kDa envelope protein (p37) that is palmitylated on cysteine residues 185186 of the 372-amino acid protein. We have previously reported on a loosely conserved consensus motif. Further analysis has identified a conserved consensus sequence, Hydro*AAC(C)A (Hydro* represents a hydrophobic portion of a protein determined by any one of the following: a hydrophobic sequence, a transmembrane domain 1-12 amino acids away from the modification site, or the prior addition of a hydrophobic molecule; C, palmitate acceptor cysteines; A, aliphatic residue) that is responsible for directing palmitylation of certain classes of palmitylproteins. We have analyzed the amino acid site occupancy upstreamdownstream of the palmitate acceptor residues in p37 by site-directed mutagenesistransient expression of mutated proteins in VV-infected cells. The two aliphatic alanines naturally found at positions 183184 of the wild-type p37 allow for efficient palmitylation. In contrast, the replacement of leucine at position 187 with glycine increases palmitylation efficiency. The 10 amino acids immediately upstream of the palmitate acceptor site are absolutely necessary while the downstream 10 amino acids are dispensable. These results together with previous data suggests that the Hydro*AAC(C)A motif is required for efficient palmitylation of p37.     

Base miscoding and strand misalignment errors by mutator Klenow polymerases with amino acid substitutions at tyrosine 766 in the O helix of the fingers subdomain

A mutant derivative of Klenow fragment DNA polymerase containing serine substituted for tyrosine at residue 766 has been shown by kinetic analysis to have an increased misinsertion rate relative to wild-type Klenow fragment, but a decreased rate of extension from the resulting mispairs (Carroll, S. S., Cowart, M., and Benkovic, S. J. (1991) Biochemistry 30, 804-813). In the present study we use an M13mp2-based fidelity assay to study the error specificity of this mutator polymerase. Despite its compromised ability to extend mispairs, the Y766S polymerase and a Y766A mutant both have elevated base substitution error rates. The magnitude of the mutator effect is mispair-specific, from no effect for some mispairs to rates elevated by 60-fold for misincorporation of TMP opposite template G. The results with the Y766S mutant are remarkably consistent with the earlier kinetic analysis of misinsertion, demonstrating that either approach can be used to identify and characterize mutator polymerases. Both the Y766S and Y766A mutant polymerases are also frameshift mutators, having elevated rates for two-base deletions and a 276-base deletion between a direct repeat sequence. However, neither mutant polymerase has an increased error rate for single-base frameshifts in repetitive sequences. This error specificity suggests that the deletions generated by the mutator polymerases are initiated by misinsertion rather than by strand slippage. When considered with recent structure-function studies of other polymerases, the data indicate that the nucleotide misinsertion and strand-slippage mechanisms for polymerization infidelity are differentially affected by changes in distinct structural elements of DNA polymerases that share similar subdomain structures.     

Ribosomal protein L9 interactions with 23 S rRNA: the use of a translational bypass assay to study the effect of amino acid substitutions

During translation of bacteriophage T4 gene 60 mRNA, ribosomes bypass 50 nucleotides with high efficiency. One of the mRNA signals for bypass is a stem-loop in the first part of the coding gap. When the length of this stem-loop is extended by 36 nucleotides, bypass is reduced to 0.35% of the wild-type level. Bypass is partially restored by a mutation in the C-terminal domain of Escherichia coli large ribosomal subunit protein L9. Previous work has shown that L9 is an elongated protein with an alpha-helix that connects and orients the N and C-terminal domains that both contain a predicted RNA binding site. We have determined two binding sites of L9 on 23 S rRNA. A 778 nucleotide RNA fragment encompassing domain V (nucleotides 1999 to 2776) of the 23 S rRNA is retained on filters by L9 and contains both sites. The N and C-terminal domains of L9 were shown to interact with nucleotides just 5' to nucleotide 2231 and 2179 of the 23 S rRNA, respectively, using the toeprint assay. These L9 binding sites on 23 S rRNA suggest that L9 functions as a brace across helix 76 to position helices 77 and 78 relative to the peptidyl transferase center. In this study, bypass on a mutant gene 60 mRNA has been used as an assay to probe the importance of particular L9 amino acids for function. Amino acid substitutions in the C-terminal domain are shown to partially restore bypass. These mutant L9 proteins have reduced binding to a 23 S rRNA fragment (nucleotides 1999 to 2274) containing domain V, to which L9 binds. They partially retain both the N and C-terminal domain interactions. On the other hand, substitutions of amino acids in the N-terminal domain, which greatly reduce RNA binding, do not restore bypass. The latter mutants have completely lost the N-terminal domain interaction. Addition of an amino acid to the alpha-helix also restores gene 60 bypass. RNA binding by this mutant is similar to that observed for the C-terminal domain mutants that partially restore bypass.     

Defects in courtship and vision caused by amino acid substitutions in a putative RNA-binding protein encoded by the no-on-transient A (nonA) gene of Drosophila

The Drosophila no-on-transient A (nonA) gene is involved in the visual behaviors and courtship song of the fly. The NONA polypeptide contains two copies of the RNA-recognition motif (RRM), a hallmark of proteins involved in RNA binding, and an adjacent conserved charged region. This 311-amino-acid region is found in four other proteins and largely overlaps the Drosophila-Behavior/Human Splicing (or DBHS) domain. The newest family member, Drosophila nAhomo, was discovered in a database search, and encodes a protein with 80% identity to NONA. In this study, three nonA mutations generated by chemical mutagenesis were sequenced and found to fall within the conserved region. Site-directed mutagenesis of the two RRMs, and within a (conserved) charged region located C-terminal to them, was performed to determine the significance of these domains with respect to whole-organismal phenotypes. Behavior and viability were assessed in transformed flies, the genetic background of which lacks the nonA locus. Point mutations of amino acid 548 in the charged region confirmed the etiology of the nonAdiss courtship-song mutation and showed that a milder substitution at this site produced intermediate singing behavior, although it failed to rescue visual defects. Mutagenesis of the RRM1 domain resulted in effects on viability, vision, and courtship song. However, amino acid substitutions in RNP-II of RRM2 led to near-normal phenotypes, and the in vivo nonA mutations located in or near RRM2 caused visual defects only. Thus, we suggest that the first RRM could be important for all functions influenced by nonA, whereas the second RRM may be required primarily for normal vision.     

Interactions of the amino acid residue at position 31 of the c-Ha-Ras protein with Raf-1 and RalGDS

The Ras and Rap1A proteins can bind to the Raf and RalGDS families. Ras and Rap1A have Glu and Lys, respectively, at position 31. In the present study, we analyzed the effects of mutating the Glu at position 31 of the c-Ha-Ras protein to Asp, Ala, Arg, and Lys on the interactions with Raf-1 and RalGDS. The Ras-binding domain (RBD) of Raf-1 binds the E31R and E31K Ras mutants less tightly than the wild-type, E31A, and E31D Ras proteins; the introduction of the positively charged Lys or Arg residue at position 31 specifically impairs the binding of Ras with the Raf-1 RBD. On the other hand, the ability of the oncogenic RasG12V protein to activate Raf-1 in HEK293 cells was only partially reduced by the E31R mutation but was drastically impaired by the E31K mutation. Correspondingly, RasG12V(E31K) as well as Rap1A, but not RasG12V(E31R), exhibited abnormally tight binding with the cysteine-rich domain of Raf-1. On the other hand, the E31A, E31R, and E31K mutations, but not the E31D mutation, enhanced the RalGDS RBD-binding activity of Ras, indicating that the negative charge at position 31 of Ras is particularly unfavorable to the interaction with the RalGDS RBD. RasG12V(E31K), RasG12V(E31A), and Rap1A stimulate the RalGDS action more efficiently than the wild-type Ras in the liposome reconstitution assay. All of these results clearly show that the sharp contrast between the characteristics of Ras and Rap1A, with respect to the interactions with Raf-1 and RalGDS, depends on their residues at position 31.     

Specific changes in the in vitro production of prostanoids by the ovine cervix at parturition

A method of tissue superfusion has been used to measure in vitro prostanoid production by the ovine cervix during late pregnancy and at parturition. In late pregnancy (105-135 days of gestation) cervical tissue produced relatively large amounts of prostaglandin E (PGE); in comparison, the production rates of prostaglandin F (PGF), 13, 14-dihydro-15-oxo-prostaglandin F (PGFM) and 6-oxo-prostaglandin F1 alpha were generally low. Thromboxane B2 (TXB2) production was minimal and often unmeasurable. There were significant increases in the production rates of PGE and 6-oxo-PGF1 alpha by cervical tissue taken immediately after delivery, when compared to late pregnancy. Mean production rates of PGE increased from 19.8 +/- 4.1 to 43.8 +/- 7.4 ng/g. dry wt./min; 6-oxo-PGF1 alpha production rates increased more than three-fold from 10.0 +/- 2.7 to 34.6 +/- 9.8 ng/g. dry wt./min (means +/- S.E.M.). There were no significant differences in the rates of production of PGF, PGFM and TXB2 by the two groups.     

WWP, a new amino acid motif present in single or multiple copies in various proteins including dystrophin and the SH3-binding Yes-associated protein YAP65

A new repeating amino acid motif, which we called WWP, was found in several proteins of yeast, nematod or vertebrate origin. Among these are dystrophin, the product of the Duchenne muscular dystrophy locus, a protein (YAP65) which associates in vitro with the Src homology domain 3 (SH3) of the Yes proto-oncogene product, and a human putative GTPase-activating protein. As is the case for proteins which contain the SH2, SH3 and PH domains, at least some of the WWP-containing proteins appear to be signaling or cytoskeletal proteins, associated with plasma or organellar membranes, and specific protein-protein contacts are likely to be crucial to their function.     

Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase

We have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C-terminus of the active-site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8,000 amino acid combinations. The most active of these 8,000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain of E. coli, DF502. Approximately 3% of the mutants complement DF502 with an activity that is above 70% of wild-type activity. The sequences of these hinge mutants reveal that the solutions to the hinge flexibility problem are varied. Moreover, these preferences are sequence dependent; that is, certain pairs occur frequently. They fall into six families of similar sequences. In addition to the hinge sequences expected on the basis of phylogenetic analysis, we selected three new families of 3-amino-acid hinges: X(A/S)(L/K/M), X(aromatic/beta-branched)(L/K), and XP(S/N). The absence of these hinge families in the more than 60 known species of triosephosphate isomerase suggests that during evolution, not all of sequence space is sampled, perhaps because there is no neutral mutation pathway to access the other families.     

Human immunodeficiency virus type 1 protease inhibitors: evaluation of resistance engendered by amino acid substitutions in the enzyme's substrate binding site

The human immunodeficiency virus type 1 (HIV-1) protease is a homodimeric aspartyl endopeptidase that is required for virus replication. A number of specific, active-site inhibitors for this enzyme have been described. Many of the inhibitors exhibit significant differences in activity against the HIV-1 and HIV type 2 (HIV-2) enzymes. An initial study was conducted to ascertain the HIV-1 protease's potential to lose sensitivity to several test inhibitors while retaining full enzymatic activity. The substrate binding sites of the HIV-1 and HIV-2 enzymes are almost fully conserved, except for four amino acid residues at positions 32, 47, 76, and 82. Accordingly, recombinant mutant type 1 proteases were constructed that contained the cognate type 2 residue at each of these four positions. The substitution at position 32 resulted in a significant adverse effect on inhibitor potency. However, this substitution also mediated a noted increase in the Km of the substrate. Individual substitutions at the remaining three positions, as well as a combination of all four substitutions, had very little effect on enzyme activity or inhibitor susceptibility. Hence, the four studied active site residues are insufficient to be responsible for differences in inhibitor sensitivity between the HIV-1 and HIV-2 proteases and are unlikely to contribute to the generation of inhibitor-resistant mutant HIV-1 protease.     

Functional roles of the conserved aromatic amino acid residues at position 108 (motif IV) and position 196 (motif VIII) in base flipping and catalysis by the N6-adenine DNA methyltransferase from Thermus aquaticus

The DNA methyltransferase (Mtase) from Thermus aquaticus (M.TaqI) catalyzes the transfer of the activated methyl group of S-adenosyl-L-methionine to the N6 position of adenine within the double-stranded DNA sequence 5'-TCGA-3'. To achieve catalysis M.TaqI flips the target adenine out of the DNA helix. On the basis of the three-dimensional structure of M.TaqI in complex with the cofactor and its structural homology to the C5-cytosine DNA Mtase from Haemophilus haemolyticus, Tyr 108 and Phe 196 were suggested to interact with the extrahelical adenine. The functional roles of these two aromatic amino acid residues in M.TaqI were investigated by mutational analysis. The obtained mutant Mtases were analyzed in an improved kinetic assay, and their ability to flip the target base was studied in a fluorescence-based assay using a duplex oligodeoxynucleotide containing the fluorescent base analogue 2-aminopurine at the target position. While the mutant Mtases containing the aromatic amino acid Trp at position 108 or 196 (Y108W and F196W) showed almost wild-type catalytic activity, the mutant Mtases with the nonaromatic amino acid Ala (Y108A and F196A) had a strongly reduced catalytic constant. Y108A was still able to flip the target base, whereas F196A was strongly impaired in base flipping. These results indicate that Phe 196 is important for stabilizing the extrahelical target adenine and suggest that Tyr 108 is involved in placing the extrahelical target base in an optimal position for methyl group transfer. Since both aromatic amino acids belong to the conserved motifs IV and XIII found in N6-adenine and N4-cytosine DNA Mtases as well as in N6-adenine RNA Mtases, a similar function of aromatic amino acid residues within these motifs is expected for the different Mtases.     

Effects of dietary protein or amino acids in the perfusion medium on amino acid metabolism in perfused adult rat liver

To elucidate the response of amino acid metabolism in the liver to dietary protein and plasma amino acids, the livers of adult rats fed on diet containing 10% (control) or 3% (low-protein) egg protein for 3 weeks were perfused for 120 min with amino acid-free medium in Experiment 1 or medium containing an amino acid mixture simulating that in plasma in Experiment 2. During perfusion about 40% of the free amino acids were lost from the liver in Exp. 1, and about 30% in Exp. 2. During this period, in Exp. 1 the releases of free amino acids and urea into the medium were 140 mumol and 2.52 mg, respectively, in the control group and 207 mumol and 1.10 mg respectively, in the low-protein group. Thus release was greater than decrease in free amino acids in the liver. Essential amino acids, particularly lysine and branched chain amino acids, were released preferentially. The results suggest that the amount of breakdown of liver protein in the two groups was similar, but that the nitrogen was mainly released as free amino acids in the low-protein group, and as urea in the control group. On the contrary, in Exp. 2 the amount of nitrogen released from the liver was comparable to the decrease in amino acids in the liver, and the releases of urea were also less, being 1.83 mg in the control group and 0.54 mg in low-protein group. The results show that amino acid metabolism in the liver is greatly affected by the nutritional state of the animal and the amino acid content of the perfusion fluid.     

An evaluation of postabsorptive protein and amino acid metabolism in the lactating dairy cow

The current protein system utilized in the US was formulated in 1985 with minor modifications in 1989 and has gained widespread acceptance. However, some of the assumptions that were adopted by the National Research Council (NRC) appear to be inconsistent with observational data. The marginal efficiency of conversion of absorbed protein to milk protein was assumed by NRC to be 70% until the requirement for absorbed protein was met and was 0% thereafter. The mean marginal efficiency observed for abomasal casein infusions reported in the literature and collected at the Purina Mills Research Center was 21%. Sorting the data into protein-sufficient and protein-deficient classes did not support the assumptions of 70% marginal efficiency in a deficient state and 0% marginal efficiency in the sufficient state. Analyses of nitrogen balance data and abomasal flow data and the work of Van Straalen et al. (77) indicated that energy status of the animal plays a role in determining the response to absorbed protein. Such a consideration was not included in the NRC model. The adoption of equations that describe metabolism at the organ level as opposed to the animal level would allow direct use of organ level data for parameterization and may provide better predictions. Simple representations of digestion and absorption, splanchnic metabolism, and mammary metabolism of amino acids or protein in aggregate are described. These representations could be used to improve the current system and could serve as a bridge to adoption of more complex models.     

Current concepts of amino acid and protein metabolism in the mammary gland of the lactating ruminant

Milk protein responses to protein nutrition are typically poor and, in part, may be due to the low efficiency (approximately 25 to 30%) of converting dietary N into milk. Posthepatic availability of amino acids (AA) is not limited, yet only approximately 30% is converted into milk. The poor capture of AA by the mammary gland may relate to the imbalanced and uncoordinated timing of nutrient delivery to the gland. The infusion of essential AA improves the efficiency of utilization (0.31); however, further catabolism of AA within the mammary gland suggests that AA transport is not a major limitation. These losses may serve ancillary or functional roles, but mammary oxidation of some AA occurs only when AA extraction exceeds the stoichiometric requirements for milk protein synthesis. Intracellular substrate supply may be more limiting than is the appartus for protein synthesis. Studies utilizing isotope labeling and conducted in vitro and in vivo now suggest that circulating peptides and proteins can serve as sources of perhaps all AA for casein synthesis, but the source of these remains elusive. Constitutive protein and casein turnover contribute significantly (42 to 72%) to mammary protein synthesis. All AA are extensively channeled through an intermediary protein pool or pools that have rapid turnover rates. The AA are then incorporated into casein, which appears to be fixed in association with protein turnover. The mammary gland is a major controller of its metabolism, and the mechanisms of AA extraction and conversion into milk protein are linked to secretion events. Blood flow may be a key point of regulation whereby mechanisms sense and respond to nutrient supply and balance to the gland via alterations in hemodynamics.