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Author(s): Shilpa Ray[sup.1] , Susmita Das[sup.1] , Pritam Kumar Panda[sup.1] and Mrutyunjay Suar[sup.1] Background Salmonellosis is one of the leading causes of mortality among infectious diseases [1]. Salmonella enterica are [...]

The dramatic increase in healthcare cost has become a significant burden to the world. Many patients are denied the accessibility of medication because of the high price of drugs. Total biosynthesis of chiral drug intermediates is an environmentally friendly approach that helps provide more affordable pharmaceuticals. Here we have expanded the natural metabolic capability to biosynthesize a nonnatural amino acid L-homoalanine, which is a chiral precursor of levetiracetam, brivaracetam, and ethambutol. We developed a selection strategy and altered the substrate specificity of ammonium-assimilating enzyme glutamate dehydrogenase. The specificity constant [k.sub.cat]/[K.sub.m] of the best mutant towards 2-ketobutyrate is 50-fold higher than that towards the natural substrate 2-ketoglutarate. Compared to transaminase livE and NADH-dependent valine dehydrogenases, the evolved glutamate dehydrogenase increased the conversion yield of 2-ketobutyrate to L-homoalanine by over 300% in aerobic condition. As a result of overexpressing the mutant glutamate dehydrogenase and Bacillus subtilis threonine dehydratase in a modified threonine-hyperproducing Escherichia coli strain (ATCC98082, [DELTA]rhtA), 5.4 g/L L-homoalanine was produced from 30 g/L glucose (0.18 g/g glucose yield, 26% of the theoretical maximum). This work opens the possibility of total biosynthesis of other nonnatural chiral compounds that could be useful pharmaceutical intermediates. chiral chemicals | directed evolution | metabolic engineering | synthetic biology doi/10.1073/pnas.0912903107

Protein synthesis involves the accurate attachment of amino acids to their matching transfer RNA (tRNA) molecules. Mistranslating the amino acids serine or glycine for alanine is prevented by the function of independent but collaborative aminoacylation and editing domains of alanyl-tRNA synthetases (AlaRSs). We show that the C-Ala domain plays a key role in AlaRS function. The C-Ala domain is universally tethered to the editing domain both in AlaRS and in many homologous free-standing editing proteins. Crystal structure and functional analyses showed that C-Ala forms an ancient single-stranded nucleic acid binding motif that promotes cooperative binding of both aminoacylation and editing domains to [tRNA.sup.Ala]. In addition, C-Ala may have played an essential role in the evolution of AlaRSs by coupling aminoacylation to editing to prevent mistranslation.

D-Alanine is a structural component of mycobacterial peptidoglycan. The primary route of D-alanine biosynthesis in eubacteria is the enantiomeric conversion from L-alanine, a reaction catalysed by D-alanine racemase (Air). Mycobacterium smegmatis air insertion mutants are not dependent on D-alanine for growth and display a metabolic pattern consistent with an alternative pathway for D-alanine biosynthesis. In this study, we demonstrate that the M. smegmatis alr insertion mutant TAM23 can synthesize D-alanine at lower levels than the parental strain. The insertional inactivation of the air gene also decreases the intracellular survival of mutant strains within primary human monocyte-derived macrophages. By complementation studies, we confirmed that the impairment of air gene function is responsible for this reduced survival. Inhibition of superoxide anion and nitric oxide formation in macrophages suppresses the differential survival. In contrast, for bacteria grown in broth, both strains had approximately the same susceptibility to hydrogen peroxide, acidified sodium nitrite, low pH and polymyxin B. In contrast, TAM23 exhibited increased resistance to lysozyme. D-Alanine supplementation considerably increased TAM23 viability in nutritionally deficient media and within macrophages. These results suggest that nutrient deprivation in phagocytic cells combined with killing mediated by reactive intermediates underlies the decreased survival of air mutants. This knowledge may be valuable in the construction of mycobacterial auxotrophic vaccine candidates.

An alanine scan of the ramoplanin A2 aglycon, a potent antibiotic that has inhibited bacterial cell wall biosynthesis, is described. The antimicrobial activity of the analogues has defined the importance of each residue of this complex glycopeptide antibiotic.

The Dlt system modulates the density of negative charge in the cell wall of Gram-positive bacteria by substituting anionic polymers (wall and lipoteichoic acids) with D-alanine. The htrA and htrB genes, regulated by the CssRS two-component system (TCS) and encoding membrane-associated protein quality control proteases, were expressed at strongly decreased levels in a mutant with defective Dlt (dltD:: miniTn 10) as compared to the [dlt.sup.+] wild-type strain under a secretion stress condition (hypersecretion of AmyQ [alpha]-amylase). The level of HtrA protein in the extracellular proteome of the dltD mutant was decreased consistently. Expression from the promoter of the lialHGFSR (yvqlHGFEC) operon ([P.sub.lial]) is dependent on the LiaRS TCS. The Dlt defect increased the expression from [P.sub.lial] under two stress conditions, AmyQ hypersecretion and treatment with a cationic antimicrobial peptide (LL-37), but decreased the expression in vancomycin-treated cells. Furthermore, Dlt inactivation enhanced the expression of the YxdJK-regulated yxdL gene in LL-37-treated cells. The increased net negative charge of the cell wall seems to cause varied and opposite effects on the expression of CssRS-, LiaRS- and YxdJK-regulated genes under different stress conditions. The results suggest that TCSs which sense misfolded proteins or peptides are modulated by the density of negative charge in the cell wall. The density of negative charge on the outer surface of the cell membrane did not have a similar effect on TCSs.

Lactobacillus plantarum produces peptidoglycan precursors ending in D-lactate instead of D-alanine, making the bacterium intrinsically resistant to vancomycin. The ligase Ddl of L. plantarum plays a central role in this specificity by synthesizing D-alanyl-D-lactate depsipeptides that are added to the precursor peptide chain by the enzyme MurF. Here we show that L. plantarum also encodes a D-Ala-D-Ala dipeptidase, Aad, which eliminates D-alanyi-D-alanine dipeptides that are produced by the Ddl ligase, thereby preventing their incorporation into the precursors. Although D-alanine-ended precursors can be incorporated into the cell wall, inactivation of Aad failed to suppress growth defects of L. plantarum mutants deficient in D-lactate-ended precursor synthesis.

Arginine utilization in Pseudomonas aeruginosa with multiple catabolic pathways represents one of the best examples of the metabolic versatility of this organism. To identify genes involved in arginine catabolism, we have employed DNA microarrays to analyze the transcriptional profiles of this organism in response to L-arginine. While most of the genes involved in arginine uptake, regulation, and metabolism have been identified as members of the ArgR (arginine-responsive regulatory protein) regulon in our previous study, they did not include any genes of the arginine dehydrogenase (ADH) pathway. In this study, 18 putative transcriptional units of 38 genes, including the two known genes of the ADH pathway, kauB and gbuA, were found to be inducible by exogenous L-arginine in the absence of ArgR. To identify the missing genes that encode enzymes for the initial steps of the ADH pathway, the potential physiological functions of those candidate genes in arginine utilization were studied by growth phenotype analysis of knockout mutants. Expression of these genes was induced by L-arginine in an aruF mutant strain devoid of a functional arginine succinyltransferase pathway, the major route of arginine utilization. Disruption of dadA, a putative catabolic alanine dehydrogenase-encoding gene, in the aruF mutant produced no growth on L-arginine, suggesting the involvement of L-alanine in arginine catabolism. This hypothesis was further supported by the detection of an L-arginineinducible arginine:pyruvate transaminase activity in the aruF mutant. Knockout of aruH and aruI, which encode an arginine:pyruvate transaminase and a 2-ketoarginine decarboxylase in an operon, also abolished the ability of the aruF mutant to grow on L-arginine. The results of high-performance liquid chromatography analysis demonstrated consumption of 2-ketoarginine and suggested that generation of 4-guanidinobutyraldehyde occurred in the aruF mutant but not in the aruF arul mutant. These results led us to propose the arginine transaminase pathway that removes the s-amino group of L-arginine via transamination instead of oxidative deamination by dehydrogenase or oxidase as originally proposed. In the same genetic locus, we also identified a two-component system, AruRS, for the regulation of arginine-responsive induction of the arginine transaminase pathway. This work depicted a wider network of arginine metabolism than we previously recognized.

The humanized monoclonal antibody HzKR127 recognizes the preS1 domain of the human hepatitis B virus surface proteins with a broadly neutralizing activity in vivo. We present the crystal structures of HzKR127 Fab and its complex with a major epitope peptide, in the complex structure, the bound peptide forms a type IV [beta]turn followed by [3.sup.10] helical turn, the looped-out conformation of which provides a structural basis for broad neutralization. Upon peptide binding, the antibody undergoes a dramatic complementarity determining region H3 lid opening. To understand the structural implication of the virus neutralization, we carried out comprehensive alanine-scanning mutagenesis of all complementarity determining region residues in HzKR127 Fab. The functional mapping of the antigen-combining site demonstrates the specific roles of major binding determinants in antigen binding, contributing to the rational design for maximal humanization and affinity maturation of the antibody. alanine-scanning mutagenesis | crystal structure | humanized antibody virus neutralization

The solution structure of the hepta-alanine polypeptide Ac[X.sub.2] [A.sub.7][O.sub.2]-N[H.sub.2] (XAO) has been a matter of controversy in the current literature. On one side of the argument is a claim that the peptide adopts a mostly polyproline II (PPII) structure, with a alanine propensity | arnide I | unfolded peptides | vibrational spectroscopy

The presence of a solvent-exposed alanine residue stabilizes a helix by 0.4-2 kcal * [mol.sup.-1] relative to glycine. Various factors have been suggested to account for the differences in helical propensity, from the higher conformational freedom of glycine sequences in the unfolded state to hydrophobic and van der Waals' stabilization of the alanine side chain in the helical state. We have performed all-atom molecular dynamics simulations with explicit solvent and exhaustive sampling of model peptides to address the backbone conformational entropy difference between Ala and Gly in the denatured state. The mutation of Ala to Gly leads to an increase in conformational entropy equivalent to [approximately equal to] 0.4 kcal 8 [mol.sup.-1] in a fully flexible denatured, that is, unfolded, state. But, this energy is closely counterbalanced by the (measured) difference in free energy of transfer of the glycine and alanine side chains from the vapor phase to water so that the unfolded alanineand glycine--containing peptides are approximately isoenergetic. The helix-stabilizing propensity of Ala relative to Gly thus mainly results from more favorable interactions of Ala in the folded helical structure. The small difference in energetics in the denatured states means that the [PHI]-values derived from Ala [right arrow] Gly scanning of helices are a very good measure of the extent of formation of structure in proteins with little residual structure in the denatured state. folding | pathway | protein | stability | transition state

D-alanine:D-alanine ligase (DDI) is an essential enzyme in bacterial cell wall biosynthesis and an important target for developing new antibiotics. It catalyzes the formation of D-alanine:D-alanine dipeptide, sequentially by using one D-alanine and one ATP as substrates for the first-half reaction, and a second D-alanine substrate to complete the reaction. Some gain of function DDI mutants can use an alternate second substrate, causing resistance to vancomycin, one of the last lines of defense against life-threatening Gram-positive infections, Here, we report the crystal structure of Staphylococcus aureus DDI (StaDDI) and its cocrystal structures with 3-chloro-2,2-dimethyl-N-[4(trifluoromethyl)phenyl]propanamide (inhibitor 1) ([K.sub.i] = 4 [micro]M against StaDDI) and with ADP, one of the reaction products, at resolutions of 2.0, 2.2, and 2.6 [angstrom], respectively. The overall structure of StaDDI can be divided into three distinct domains. The inhibitor binds to a hydrophobic pocket at the interface of the first and the third domain. This inhibitor-binding pocket is adjacent to the first D-alanine substrate site but does not overlap with any substrate sites. An allosteric inhibition mechanism of StaDDI by this compound was proposed. The mechanism provides the basis for developing new antibiotics targeting D-alanine:D-alanine ligase. Because this compound only interacts with residues from the first D-alanine site, inhibitors with this binding mode potentially could overcome vancomycin resistance. antibiotic | crystallography | inhibition mechanism | kinetics | cell wall synthesis

It has been shown previously that expression of the Streptomyces lividans clpP1P2 operon, encoding proteolytic subunits of the Clp complex, the clpC1 gene, encoding the ATPase subunit, and the Ion gene, encoding another ATP-dependent protease, are all activated by ClgR. The ClgR regulon also includes the clgR gene itself. It is shown here that the degradation of ClgR and Lon is ClpP1/P2-dependent and that the two C-terminal alanines of these new substrates are involved in their stability. The ClpC1 protein, which does not end with two alanines, is also accumulated in a clpP1P2 mutant. The results presented here support the idea that ClpP1/P2 ensure post-translational control of ClgR regulon members, including ClgR itself.

CS1 pili are important virulence factors of enterotoxigenic Escherichia coli strains associated with human diarrheal disease. They are the prototype for a family of pili that share extensive sequence similarity among their structural and assembly proteins. Only four linked genes, cooB, cooA, cooC, and cooD, are required to produce CSI pili in E. coli K-12. To identify amino acids important for the function of the major pilin CooA, we used alanine substitution mutagenesis targeting conserved residues in the N and C termini of the protein. To test function, we examined cooA mutants for the ability to agglutinate bovine erythrocytes. Each hemagglutination-negative (H[A.sup.-]) cooA mutant was examined to identify its assembly pathway defect. CooA has been shown to be degraded in the absence of CooB (K. Voegele, H. Sakellaris, and J. R. Scott, Proc. Natl. Acad. Sci. USA 94:13257-13261, 1997). We found several H[A.sup.-] cooA mutants that produced no detectable CooA, suggesting that recognition by CooB is mediated by residues in both the N and C termini of CooA. In addition, we found that alanine substitution for some of the conserved residues in the C-terminal motif 'AGxYxG([x.sub.6])T,' which is found in all subunits of this pilus family, had no effect on pilus formation. However, alanine substitution for some of the alternating hydrophobic residues within this motif prevented CooA from interacting with CooD, which serves as both the tip adhesin and nucleation protein for pilus formation. Thus, it appears that some, but not all, of the residues in both the N and C termini of CooA play a critical role in the intermolecular interactions of the major pilin with the other structural and assembly proteins. We anticipate that the results obtained here for CS1 pili in enterotoxigenic E. coli will help develop an understanding of the pilus assembly pathway used by CS1 family members in several important human pathogens.

As a general rule, ribosomally synthesized polypeptides contain amino acids only in the L-isoform in an order dictated by the coding DNA/RNA. Two of a total of only four examples of L to O conversions in prokaryotic systems occur in posttranslationally modified antimicrobial peptides called lantibiotics. In both examples (lactocin S and lacticin 3147), ribosomally encoded L-serines are enzymatically converted to D-alanines, giving rise to an apparent mistranslation of serine codons to alanine residues. It has been suggested that this conversion results from a two-step reaction initiated by a lantibiotic synthetase converting the gene-encoded L-serine to dehydroalanine (dha). By using lacticin 3147 as a model system, we report the identification of an enzyme, LtnJ, that is responsible for the conversion of dha to D-alanine. Deletion of this enzyme results in the residues remaining as dha intermediates, leading to a dramatic reduction in the antimicrobial activity of the producing strain. The importance of the chirality of the three D-alanines present in lacticin 3147 was confirmed when these residues were systematically substituted by L-alanines. In addition, substitution with L-threonine (ultimately modified to dehydrobutyrine), glycine, or L-valine also resulted in diminished peptide production and/or relative activity, the extent of which depended on the chirality of the newly incorporated amino acid(s). antimicrobial | bacteriocin | chirality

AlaX is the homologue of the class II alanyl-tRNA synthetase editing domain and has been shown to exhibit autonomous editing activity against mischarged tRN[A.sup.Ala]. Here, we present the structures of AlaX from the archaeon Pyrococcus horikoshii in apo form, complexed with zinc, and with noncognate amino acid L-serine and zinc. Together with mutational analysis, we demonstrated that the conserved Thf-30 hydroxyl group located near the [beta]-methylene of the bound serine is responsible for the discrimination of noncognate serine from cognate alanine, based on their chemical natures. Furthermore, we confirmed that the conserved Gin-584 in alanyl-tRNA synthetase, which corresponds to Thr-30 of AlaX, is also critical for discrimination. These observations strongly suggested conservation of the chemical discrimination among trans- and cis-editing of tRN[A.sup.Ala]. alanyl-tRNA synthetase | class II tRNA synthetase | crystal structure | trans-editing

Among the archaea, Methanococcus maripaludis has the unusual ability to use L- or D-alanine as a nitrogen source. To understand how this occurs, we tested the roles of three adjacent genes encoding homologs of alanine dehydrogenase, alanine racemase, and alanine permease. To produce mutations in these genes, we devised a method for markerless mutagenesis that builds on previously established genetic tools for M. maripaludis. The technique uses a negative selection strategy that takes advantage of the ability of the M. maripaludis hpt gene encoding hypoxanthine phosphoribosyltransferase to confer sensitivity to the base analog 8-azahypoxanthine. In addition, we developed a negative selection method to stably incorporate constructs into the genome at the site of the upt gene encoding uracil phosphoribosyltransferase. Mutants with in-frame deletion mutations in the genes for alanine dehydrogenase and alanine permease lost the ability to grow on either isomer of alanine, while a mutant with an in-frame deletion mutation in the gene for alanine racemase lost only the ability to grow on D-alanine. The wild-type gene for alanine dehydrogenase, incorporated into the upt site, complemented the alanine dehydrogenase mutation. Hence, the permease is required for the transport of either isomer, the dehydrogenase is specific for the e isomer, and the raeemase converts the o isomer to the L isomer. Phylogenetic analysis indicated that all three genes had been acquired by lateral gene transfer from the low-moles-percent G+C gram-positive bacteria.

The observation that, on a molar basis, guanidinium chloride (GdmCl) is 2-2.5-fold more effective as a protein denaturant than urea is reinvestigated using an alanine-based helical peptide (alahel) that lacks side chain electrostatic contributions to stability. It is found that GdmCl is only slightly more powerful as a denaturant of alahel than urea in salt-free buffer.

A novel alanine dehydrogenase (AlaDH) showing no significant amino acid sequence homology with previously known bacterial AlaDHs was purified to homogeneity from the soluble fraction of the hyperthermophilic archaeon Archaeoglobus fulgidus. AlaDH catalyzed the reversible, NA[D.sup.+]-dependent deamination of L-alanine to pyruvate and N[H.sub.4.sup.+]. NADP(H) did not serve as a coenzyme. The enzyme is a homodimer of 35 kDa per subunit. The [K.sub.m] values for L-alanine, NA[D.sup.+], pyruvate, NADH, and N[H.sub.4.sup.+] were estimated at 0.71, 0.60, 0.16, 0.02, and 17.3 mM, respectively. The A. fulgidus enzyme exhibited its highest activity at about 82[degrees]C (203 U/mg for reductive amination of pyruvate) yet still retained 30% of its maximum activity at 25[degrees]C. The thermostability of A. fulgidus AlaDH was increased by more than 10-fold by 1.5 M KCl to a half-life of 55 h at 90[degrees]C. At 25[degrees]C in the presence of this salt solution, the enzyme was ~100% stable for more than 3 months. Closely related A. fulgidus AlaDH homologues were found in other archaea. On the basis of its amino acid sequence, A. fulgidus AlaDH is a member of the ornithine cyclodeaminase-[mu]-crystallin family of enzymes. Similar to the [mu]-crystallins, A. fulgidus AlaDH did not exhibit any ornithine cyclodeaminase activity. The recombinant human [mu]-crystallin was assayed for AlaDH activity, but no activity was detected. The novel A. fulgidus gene encoding AlaDH, AF1665, is designated ala.

The finding that short alanine peptides possess a high fraction of polyproline II (PII) structure ([PHI] = -75[degrees], [PSI] = +145[degrees]) at low temperature has broad implications for unfolded states of proteins. An important question concerns whether or not this structure is locally determined or cooperative. We have monitored the conformation of alanine in a series of model peptides AcGG(A)nGGN[H.sub.2] (n = 1-3) over a temperature range from -10[degrees]C to +80[degrees]C. Use of [sup.15]N-labeled alanine substitutions makes it possible to measure [sup.3][J.sub.[alpha]N] coupling constants accurately over the full temperature range. Based on a 1D next-neighbor model, the cooperative parameter [sigma] of PII nucleation is evaluated from the coupling constant data. The finding that [sigma] is close to unity (1 [+ or -] 0.2) indicates a noncooperative role for alanine in PII structure formation, consistent with statistical surveys of the Protein Data Bank that suggest that most PII structure occurs in isolated residues. Lack of cooperativity in these models implies that hydration effects that influence PII conformation in water are highly localized. Using a nuclear Overhauser effect ratio strategy to define the alanine [PSI] angle, we estimate that, at 40[degrees]C, the time-averaged alanine conformation ([PHI] = -80[degrees], [PSI] = +170[degrees]) deviates from canonical PII structure, indicating that PII melts at high temperature. Thus, the high-temperature state of short alanine peptides seems to be an unfolded ensemble with higher distribution in the extended [beta] structure basin, but not a coil.

The helix-coil transition kinetics of a series of Ala-based alpha helical peptides of different lengths with and without end caps are examined by using time-resolved infrared spectroscopy coupled with laser-induced temperature jump initiation method. The findings reveal that efficient end-capping sequences can not only stabilize preexisting helices but also promote helix formation through initiation.

The use of vibrational spectroscopy to determine the structure of tripeptides in solution is demonstrated and it is showed that alanine based tripeptides adopt stable extended structures assignable to the upper left square of the Ramachandran plot. Hydrogen bonding between the peptide and water molecules contributes to the inhomogeneous broadening of amide I bands and stabilizes the polyproline II conformation.

Radiation-induced free radical formation in the amino acid l-alpha-alanine is studied using powder and single-crystal X-, K-, and Q-band electron paramagnetic resonance (EPR) spectroscopy, X-band powder electron-nuclear double resonance (ENDOR), thermal annealing, and EPR spectrum simulations.

Research into the dihedral angles of tripeptides is presented. It was found that trialanine has a 3(sub 1)-helical structure in D2O for all of its three protonation states.

Research into the helix-coil transition of a synthetic alpha-helical peptide, Ac-YGG(KAAAA)3-CO-D-Arg-CONH2, is presented. The multiexponential relaxation kinetics indicate that the helix-coil transition is not a two-state process.

The results of improving the OPLS-AA force field for peptides by means of refitting the key Fourier torsional coefficients are discussed. A novel approach to fitting torsional parameters for electrostatically charged molecular systems is discussed, and tested on five dipeptides with charged side chains.

Chemistry research is presented describing the study and development of a model to analyse alanine racemase for enzyme-substrate interactions.

Research has been conducted on the vancomycin resistance in Enterococcus gallinarum. The investigation of the racemization selectivity of L-Ser or L-Ala by a membrane-bound serine racemase is discussed.

Research has been conducted on the tubule-based motor protein kinesisn. The experiments with alanine mutations in solvent-exposed residues are described.

Research has been conducted on lysine 73. Mutation of lysine 73 to alanine in the beta-lactamase has been investigated.

Research has been conducted on the site-directed mutagenesis. The use of this mutagenesis in replacing alanine 305 with phenylalanine and serine in cytochrome P450 3A4 active site is described.

An new study investigates the conformational behaviour of beta-alanine zwitterion in aqueous solution.

A new study uses high-resolution magic angle spinning NMR to investigate atomic detail in the resin-bound aggrecan sequence.

Research has been conducted on the aromatic to recombinant murine interleukine-6 (mIL-6) alanine mutants. The role of these mutants in mIL-6 structure and function is analyzed.

Research has been conducted on the C2-domain of the human factor V. Amino acid function within the C2-domain has been investigated using charge to alanine mutagenesis.

Research has been conducted on the Apo2L structure at 1.3 angstrom resolution. Results include the use of alanine-scanning mutagenesis for receptor contact regions mapping.

Issues concerning aminyl radicals as important transients in the hydroxyl radical-induced oxidation of the anions of alpha-methylalanine, glycine and alanine are presented.

Research has been conducted on the gene (aldA) from Myxococcus xanthus. Results indicate that this gene encodes functional alanine dehydrogenase.

Cysteinyl residues have been altered to alanine or serine and double mutant constructs have been made. Results indicate that redox couple is responsible for the arsenate electron donation.

Research was conducted to determine the amino acid residues involved in the binding and toxicity properties of the Cry1Ac toxin using alanine substituted mutants. Results show that binding affinity relative to toxicity was affected which depended on the particular residue that was being substituted.

The cloning and expression in Escherichia coli of the genes encoding NAD+-dependent alanine dehydrogenases (AlaDHs) (EC1.4.1.1) from the Antarctic bacterial organisms Shewanella sp strain Ac10 (SheAlaDH) and Carnobacterium sp strain St2 (CarAlaDH) are described. The highest level of sequence similarity to the AlaDH from the gram-negative bacterium Vibrio pproteolyticus was observed in SheAlaDH. Results indicate that the thermal instability of SheAlaDH and CarAlaDH may be caused by the relatively low numbers of salt bridges in the proteins.

Research was conducted to purify and characterize the mutants of tyrosine hydroxylase (TyrH) with alanine substituted for Phe300 or Phe309. The structures of the catalytic domain of TyrH with and without dihydropterin bound have identified active-site residues that may have a role to play in the catalytic mechanism. Complementary evidence regarding the roles of various residues is provided by site-directed mutagenesis.

Alanine-scanning site-directed mutagenesis of ionizable neutral acids that are conserved for all malic enzymes available were used to map the active site topography of the NAD-malic enzyme from Ascaris suum. Each of the conserved ionizable neutral residues were altered to alanine to examine their individual function. Results indicate that it is a possible candidate for the general base that accepts a proton from the malate hydroxyl in the oxidation step.

Escherichia coli is a significant indicator of fecal contamination in foods. Alanine racemase is required for biosynthesis of bacterial cell walls and is a typical prokaryotic enzyme. E. coli strains have genes for this enzyme and it may be useful as an indicator to detect E. coli in foods. The specificity of oligonucleotide primers from conserved sequences of the E. coli alr gene for detecting E. coli in foods, was determined.

The rotational transitions of seven isotopomers of alaninamide, the amide derivative of alanine, were measured by employing a Fourier transform microwave spectrometer. Least-squares fitting and Kraitchman's technique of isotopic substitution were utilized to determine the heavy-atom structure and indicate that the spectra can be assigned to a conformation with an intramolecular hydrogen bond from the amide to the amine.

The geometries and hyperfine coupling constants of the radiation products of L-alpha-alanine at 295 K, namely, radicals R1, *CH(CH3)COOH; R2, H3N+C*(CH3)COO(super -1); and R3, H2NC*(CH3)COOH, were studied using density functional theory. Four conformers were noted for R1 and R3, with the relative energies of each showing that structures with intramolecular hydrogen bonding are more stable. Meanwhile, a planar structure of R2 in a zwitterionic form was derived using the Onsager model.

A study was conducted to analyze an alanine dipeptide molecule in aqueous solution using molecular dynamics computer simulations. The structural and dynamical characteristics of the hydration water molecules were computed separately for solvent molecules surrounding different atoms or atomic groups. Results indicated differences between the properties of water around the polar and nonpolar atomic groups.

Research was conducted to examine the role of the three conserved glycine residues in the reactive thiol region of Dictyostelium discoideum myosin II. A combined molecular genetic and kinetic approach was employed to analyze the effect of mutations G680A, G684A and G691A on the actin binding, nucleotide binding and communication between nucleotide and actin binding sites of the myosin head fragment. Results indicate that replacing G6800 with alanine generates a long-lived actomyosin*ADP complex.

It has been reported that substitution of [Arg.sup.258], a residue within the GTPase domain of the heterotrimeric guanine nucleotide binding protein (G protein) [Alpha]-sub-unit ([[Alpha].sub.s]), to alanine ([[Alpha].sub.s]-R258A) results in decreased activation by receptor or aluminum fluoride (Al[[F.sub.4].sup.-]) and increased basal GDP release. [Arg.sup.258] interacts with [Gln.sup.170] in the helical domain, and, presumably, loss of this interaction between the GTPase and helical domain leads to more rapid GDP release, resulting in decreased activation by Al[[F.sub.4].sup.-] and increased thermolability. In this study, we mutate [Gin.sup.170] to alanine ([[Alpha].sub.s]-Q170A) and demonstrate that this mutant, like [[Alpha].sub.s]-R258A, has decreased activation by Al[[F.sub.4].sup.-], increased thermolability (both reversed in the presence of excess guanine nucleotide), and an increased rate of GDP release. However, unlike [[Alpha].sub.s]R258A, [[Alpha].sub.s]-Q170A does not have impaired receptor-mediated activation. Therefore, this interdomain interaction is critical to maintain normal guanine nucleotide binding (and hence normal activation by Al[[F.sub.4].sup.-]) but is not important for receptor-mediated activation. In single turnover GTPase assays, the catalytic rate for GTP hydrolysis of [[Alpha].sub.s]-R258A was 14-fold higher than normal whereas that of [[Alpha].sub.s]-Q170A was unaffected. Examination of the [[Alpha].sub.s] crystal structure suggests that [Arg.sup.258], through interactions with [Glu.sup.50], might constrain the position of [Arg.sup.201], a residue critical for catalyzing the GTPase reaction. This is an example of a mutation in a heterotrimeric G protein that results in an increased intrinsic GTPase activity and provides another mechanism by which G protein mutations can impair signal transduction.

Alanine-based peptides of defined sequence and length show measurable helix contents, allowing them to be used as a model system both for analyzing the mechanism of helix formation and for investigating the contributions of side-chain interactions to protein stability. Extensive characterization of many peptide sequences with varying amino acid contents indicates that the favorable helicity of alanine-based peptides can be attributed to the large helix-stabilizing propensity of alanine. based on their analysis of alanine-rich sequences N-terminally linked to a synthetic helix-inducing template, Kemp and coworkers [Kemp, D. S., Boyd, J. G. & Muendel, C. C. (1991) Nature (London) 352, 451-454; Kemp, D. S., Oslick, S. L. & Allen, T. J. (1996) J. Am. Chem. Soc. 118, 4249-4255] argue that alanine is helix-indifferent, however, and that the favorable helix contents of alanine-based peptides must have some other explanation. Here, we show that the helix contents of template-nucleated sequences are influenced strongly by properties of the template-helix j unction. A model in which the helix propensities of residues at the template-peptide junction are treated separately brings the results from alanine-based peptides and template-nucleated helices into agreement. The resulting model provides a physically plausible resolution of the discrepancies between the two systems and allows the helix contents of both template-nucleated and standard peptide helices to be predicted by using a single set of helix propensities. Helix formation in both standard peptides and template-peptide conjugates can be attributed to the large intrinsic helix-forming tendency of alanine.