Structural and functional studies of Bacillus stearothermophilus serine hydroxymethyl transferase: the role of N341, Y60 and F351 in tetrahydrofolate binding.

SHMT (serine hydoxymethyltransferase), a type I pyridoxal 5'-phosphate-dependent enzyme, catalyses the conversion of L-serine and THF (tetrahydrofolate) into glycine and 5,10 -methylene THE SHMT also catalyses several THF-independent side reactions such as cleavage of P-hydroxy amino acids, trans-amination, racemization and decarboxylation. In the present study, the residues Asn(341), Tyr(60) and Phe(351), which are likely to influence THF binding, were mutated to alanine, alanine and glycine respectively, to elucidate the role of these residues in THF-dependent and -independent reactions catalysed by SHMT. The N341A and Y60A bsSHMT (Bacillus stearothermophilus SHMT) mutants were inactive for the THF-dependent activity, while the mutations had no effect on THF-independent activity. However, mutation of Phe(351) to glycine did not have any effect oil either of the activities. The crystal structures of the glycine binary complexes of the mutants showed that N341A bsSHMT forms an external aldimine as in bsSHMT, whereas Y60A and F351G bsSHMTs exist as a Mixture of internal/external aldimine and gem-diamine forms. Crystal structures of all of the three Mutants obtained in the presence of L-allo-threonine were similar to the respective glycine binary complexes. The structure of the ternary complex of F351G bsSHMT with glycine and FTHF (5-formyl THF) showed that the monoglutamate side chain of FTHF is ordered in both the subunits of the asymmetric unit, unlike in the wild-type bsSHMT. The present studies demonstrate that the residues Asn(341) and Tyr(60) are pivotal for the binding of THF/FTHF, whereas Phe(351) is responsible for the asymmetric binding of FTHF in the two subunits of the dimer.

Crystal structures of Salmonella typhimurium propionate kinase and its complex with Ap4A: evidence for a novel Ap4A synthetic activity

Propionate kinase catalyses the last step in the anaerobic breakdown of L-threonine to propionate in which propionyl phosphate and ADP are converted to propionate and ATR Here we report the structures of propionate kinase (TdcD) in the native form as well as in complex with diadenosine 5 ',5 '''-P-1,P-4-tetraphosphate (AP(4)A) by X-ray crystallography. Structure of TdcD obtained after cocrystallization with ATP showed Ap(4)A bound to the active site pocket suggesting the presence of Ap(4)A synthetic activity in TdcD. Binding of Ap(4)A to the enzyme was confirmed by the structure determination of a TdcD-Ap(4)A complex obtained after cocrystallization of TdcD with commercially available Ap(4)A. Mass spectroscopic studies provided further evidence for the formation of Ap(4)A by propionate kinase in the presence of ATP. In the TdcD-Ap(4)A complex structure, Ap(4)A is present in an extended conformation with one adenosine moiety present in the nucleotide binding site and other in the proposed propionate binding site. These observations tend to support direct in-line transfer of phosphoryl group during the kinase reaction.

Structure of the putative mutarotase YeaD from Salmonella typhimurium in two different forms: in vivo binding of a sugar phosphate

Salmonella typhimurium YeaD (stYeaD), annotated as a putative aldose 1-epimerase, has a very low sequence identity to other well characterized mutarotases. Sequence analysis suggested that the catalytic residues and a few of the substrate-binding residues of galactose mutarotases (GalMs) are conserved in stYeaD. Determination of the crystal structure of stYeaD in an orthorhombic form at 1.9 angstrom resolution and in a monoclinic form at 2.5 angstrom resolution revealed this protein to adopt the beta-sandwich fold similar to GalMs. Structural comparison of stYeaD with GalMs has permitted the identification of residues involved in catalysis and substrate binding. In spite of the similar fold and conservation of catalytic residues, minor but significant differences were observed in the substrate- binding pocket. These analyses pointed out the possible role of Arg74 and Arg99, found only in YeaD-like proteins, in ligand anchoring and suggested that the specificity of stYeaD may be distinct from those of GalMs

Cloning, expression, purification and preliminary X-ray crystallographic studies of 2-methylisocitrate lyase from Salmonella typhimurium. Acta Crystallogr

In Salmonella typhimurium, propionate is oxidized to pyruvate via the 2-methylcitric acid cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate, is catalysed by 2-methylisocitrate lyase (EC 4.1.3.30). Methylisocitrate lyase (molecular weight 32 kDa) with a C-terminal polyhistidine affinity tag has been cloned and overexpressed in Escherichia coli and purified and crystallized under different conditions using the hanging-drop vapour-diffusion technique. Crystals belong to the orthogonal space group P2(1)2(1)2(1), with unit-cell parameters a = 63.600, b = 100.670, c = 204.745 Angstrom. A complete data set to 2.5 Angstrom resolution has been collected using an image-plate detector system mounted on a rotating-anode X-ray generator.

Studies on simultaneous nhibition of trypsin and chymotrypsin by horsegram Bowman-Birk inhibitor

Bowman-Birk inhibitors (BBI) isolated from plant seeds are small proteins active against trypsin and/or chymotrypsin. These inhibitors have been extensively studied in terms of their structure, interactions, function and evolution. Examination of the known three-dimensional structures of BBIs revealed similarities and subtle differences.The hydrophobic core, deduced from surface accessibility and hydrophobicity plots, corresponding to the two tandem structural domains of the double headed BBI are related by an almost exact two-fold, in contrast to the reactive site loops which depart appreciably from the two-fold symmetry. Also, the orientations of inhibitory loops in soybean and peanut inhibitors were different with respect to the rigid core. Based on the structure of Adzuki bean BBI-trypsin complex, models of trypsin and chymotryspin bound to the monomeric soybean BBI (SBI) were constructed. There were minor short contacts between the two enzymes bound to the inhibitor suggesting near independence of binding. Binding studies revealed that the inhibition of one enzyme in the presence of the other is associated with a minor negative cooperativity. In order to assess the functional significance of the reported oligomeric forms of BBI, binding of proteases to the crystallographic and non-crystallographic dimers as found in the crystal structure of peanut inhibitor were examined. It was found that all the active sites in these oligomers cannot simultaneously participate in inhibition.

Source and target enzyme signature in serine protease inhibitor active site sequences

Amino acid sequences of proteinaceous proteinase inhibitors have been extensively analysed for deriving information regarding the molecular evolution and functional relationship of these proteins. These sequences have been grouped into several well defined families. It was found that the phylogeny constructed with the sequences corresponding to the exposed loop responsible for inhibition has several branches that resemble those obtained from comparisons using the entire sequence. The major branches of the unrooted tree corresponded to the families to which the inhibitors belonged. Further branching is related to the enzyme specificity of the inhibitor. Examination of the active site loop sequences of trypsin inhibitors revealed that there are strong preferences for specific amino acids at different positions of the loop. These preferences are inhibitor class specific. Inhibitors active against more than one enzyme occur within a class and confirm to class specific sequence in their loops. Hence, only a few positions in the loop seem to determine the specificity. The ability to inhibit the same enzyme by inhibitors that belong to different classes appears to be a result of convergent evolution

Crystal structure of cadaverine dihydrochloride monohydrate

The structure of cadaverine dihydrochloride monohydrate has been determined by X-ray crystallography with the following features: NH3+(CH2)5NH3+.2Cl-.H2O, formula weight 191.1, monoclinic, P2, a = 11.814(2) angstrom, b = 4.517(2) angstrom, c = 20.370(3) angstrom, beta = 106.56-degrees(1): V = 1041.9(2) angstrom3, lambda = 1.541 angstrom; mu = 53.4 1; T = 296-degrees; Z = 4, D(x) = 1.218 g.cm-3, R = 0.101 for 1383 observed reflections. The crystal is highly pseudosymmetric with 2 molecules of cadaverine, 4 chloride ions and 2 partially disordered water molecules present in the asymmetric unit. Though both the cadaverine molecules in the asymmetric unit have an all trans conformation, the carbon backbones are slightly bent. Between the concave surfaces of two bent cadaverine molecules exists water channels all along the short b axis. The water molecules present in the channels are partially disordered

Crystal structures of propanediamine complexed with L and DL- glutamic acid: effect of chirality on propanediamine.

The structures of complexes of 1,3-diaminopropane With L- and DL-glutamic acid have been determined. L-Glutamic acid complex: C3H12N22+.2C5H8NO4-, M(r) = 368.4, orthorhombic. P2(1)2(1)2(1), a = 5.199 (1), b = 16.832 (1). c = 20.076 (3) angstrom, V = 1756.6 (4) angstrom3, z = 4, D(x) = 1.39 g cm-3, lambda(Mo K-alpha) = 0.7107 angstrom, mu = 1.1 cm-1, F(000) = 792. T = 296 K, R = 0.044 for 1276 observed reflections. DL-Glutamic acid complex: C3H12N22+.2C5H8NO4-, M(r) = 368.4, orthorhombic, Pna2(1), a = 15.219(2), b = 5.169 (1), c 22.457 (4) angstrom, V = 1766.6 (5) angstrom3 Z = 4, D(x) = 1.38 g cm-3, lambda(Mo K-alpha) = 0.7107 angstrom, mu = 1.1 cm F(000) = 792, T = 296 K, R = 0.056 for 993 observed reflections. The conformation of diaminopropane is all-trans in the DL complex but trans-gauche in the L complex. The main packing feature in the L complex is the arrangement of diaminopropane around dimers of antiparallel L-glutamic acid molecules. The diaminopropane in the DL complex is sandwiched between two antiparallel glutamic acid molecules of the same chirality and this forms the basic packing unit. This might be the dominant form of interaction between L-glutamic acid and diaminopropane in solution. The structures reveal the adaptability of the polyamine backbone to different environments and the probable reasons for their choice as biological cations.

Nucleotide sequence of the 3terminal region of belladonna mottle virus (renamed Physalis mottle virus) RNA and analysis of the evolutionary relationships among tymoviral coat proteins

The 3' terminal 1255 nt sequence of Physalis mottle virus (PhMV) genomic RNA has been determined from a set of overlapping cDNA clones. The open reading frame (ORF) at the 3' terminus corresponds to the amino acid sequence of the coat protein (CP) determined earlier except for the absence of the dipeptide, Lys-Leu, at position 110-111. In addiition, the sequence upstream of the CP gene contains the message coding for 178 amino acid residues of the C-terminus of the putative replicase protein (RP). The sequence downstream of the CP gene contains an untranslated region whose terminal 80 nucleotides can be folded into a characteristic tRNA-like structure. A phylogenetic tree constructed after aligning separately the sequence of the CP, the replicase protein (RP) and the tRNA-like structure determined in this study with the corresponding sequences of other tymoviruses shows that PhMV wrongly named belladonna mottle virus [BDMV(I)] is a separate tymovirus and not another strain of BDMV(E) as originally envisaged. The phylogenetic tree in all the three cases is identical showing that any subset of genomic sequence of sufficient length can be used for establishing evolutionary relationships among tymoviruses.

Rigid and flexible region in lysozyme and the invariant features in its hydration shell

Water-mediated transformations provide a useful handle for exploring the flexibility in protein molecules and the invariant features in their hydration shells. Low-humidity monoclinic hen egg white lysozyme, resulting from such a transformation, has perhaps the lowest solvent content observed in any protein crystal so far and has a well-ordered structure. A detailed comparison involving this structure, low-humidity tetragonal lysozyme, and the other available refined crystal structures of the enzyme permits the delineation of the relatively rigid, moderately flexible and highly flexible regions of the molecule. The relatively rigid region forms a contiguous structural unit close to the molecular centroid and encompasses parts of of the main beta-structure and three alpha-helices. The hydration shell of the protein contains 30 invariant water molecules. Many of them are involved in holding different parts of the molecule together or in stabilizing local structure. Five of the six invariant water molecules attached to the substrate-binding region form part of a water cluster contiguous with the side-chains of the catalytic residues Glu-35 and Asp-52.

Psi-angle dependence of geminal proton–carbonyl-13C coupling constants in peptides. A theoretical investigation†

Theoretical calculations of the geminal carbonyl-13C- proton coupling constant, 2J(C′H), in α-amino acids have been carried out using Dirac Vector model and Penney-Dirac bond order formulations. The results indicate that the couplings are dependent on the backbone torsion angle psi (ψ) of the amino acid residues in peptides. The meagre available experimental data seem to support the theoretical findings.

Structure-function relationships of icosahedral plant viruses

X-ray diffraction studies on single crystals of a few viruses have led to the elucidation of their three dimensional structure at near atomic resolution. Both the tertiary structure of the coat protein subunit and the quaternary morganization of the icosahedral capsid in these viruses are remarkably similar. These studies have led to a critical re-examination of the structural principles in the architecture of isometric viruses and suggestions of alternative mechanisms of assembly. Apart from their role in the assembly of the virus particle, the coat proteins of certian viruses have been shown to inhibit the replication of the cognate RNA leading to cross-protection. The coat protein amino acid sequence and the genomic sequence of several spherical plant RNA viruses have been determined in the last decade. Experimental data on the mechanisms of uncoating, gene expression and replication of several classes of viruses have also become available. The function of the non-structural proteins of some viruses have been determined. This rapid progress has provided a wealth of information on several key steps in the life cycle of RNA viruses. The function of the viral coat protein, capsid architecture, assembly and disassembly and replication of isometric RNA plant viruses are discussed in the light of this accumulated knowledge.