Mode of interaction of aminooxy compounds with sheep liver serine hydroxymethyltransferase

The interaction of aminooxy compounds such as aminooxyacetate (AAA), L-canaline, and hydroxylamine with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) was studied by absorption spectra and stopped-flow spectrophotometry and compared with the unique feature of interaction of O-amino-D-serine (OADS) with the enzyme [Baskaran, N., Prakash, V., Appu Rao, A. G., Radhakrishnan, A. N., Savithri, H. S., & Appaji Rao, N. (1989) Biochemistry (preceding paper in this issue)]. The reaction of AAA (0.5 mM) with the Schiff base of the enzyme resulted in the formation of pyridoxal 5'-phosphate (PLP) and was biphasic with rate constants of 191 and 19 s-1. The formation of the PLP-AAA oxime measured by decrease in absorbance at 388 nm on interaction of AAA with the enzyme had a rate constant of 5.2 M-1 s-1. On the other hand, the reaction of L-canaline with the enzyme was slower as measured by the disruption of enzyme-Schiff base than the reaction of OADS and AAA. In contrast, the formation of PLP as an intermediate could not be detected upon the interaction of hydroxylamine with the enzyme. The reaction of D-cycloserine with the enzyme was much slower (1.6 x 10(2) M-1 s-1) than the aminooxy compounds. These observations indicate that the aminooxy compounds that are structural analogues of serine (OADS, AAA, and canaline) formed PLP as an intermediate prior to the formation of oxime, whereas with hydroxylamine such an intermediate could not be detected.     

Production of biosurfactant “Biosur-Pm” by Pseudomonas maltophila CSV89: characterization and role in hydrocarbon uptake

Pseudomonas maltophilia CSV89, a soil bacterium, produces an extracellular biosurfactant, Biosur-Pm. The partially purified product is nondialyzable and chemically composed of 50% protein and 12–15% sugar, which indicates the complex nature of Biosur-Pm. It reduces the surface tension of water from 73 to 53×10^-3 N m^-1 and has a critical micellar concentration of 80 mg/l. Compared to aliphatic hydrocarbons, Biosur-Pm shows good activity against aromatic hydrocarbons. The emulsion formed is stable and does not require any metal ions for emulsification. The kinetics of Biosur-Pm production suggest that its synthesis is a growth-associated and pH-dependent process. At pH 7.0, cells produced more Biosur-Pm with less cell surface hydrophobicity. At pH 8.0, however, the cells produced less Biosur-Pm with more cell surface hydrophobicity and showed a twofold higher affinity for aromatic hydrocarbons compared to the cells grown at pH 7.0. The Biosur-Pm showed a pH-dependent release, stimulated growth of the producer strain on mineral salts medium with 1-naphthoic acid when added externally, and facilitated the conversion of salicylate to catechol. All these results suggest that Biosur-Pm is probably a cell-wall component and helps in hydrocarbon assimilation/uptake.     

Overexpression of yeast spermidine synthase impacts ripening,senescence and decay symptoms in tomato

Polyamines (PAs) are ubiquitous, polycationic biogenic amines that are implicated in many biological processes, including plant growth and development, but their precise roles remain to be determined. Most of the previous studies have involved three biogenic amines: putrescine (Put), spermidine (Spd) and spermine (Spm), and their derivatives. We have expressed a yeast spermidine synthase (ySpdSyn) gene under constitutive (CaMV35S) and fruit‐ripening specific (E8) promoters in Solanum lycopersicum (tomato), and determined alterations in tomato vegetative and fruit physiology in transformed lines compared with the control. Constitutive expression of ySpdSyn enhanced intracellular levels of Spd in the leaf, and transiently during fruit development, whereas E8‐ySpdSyn expression led to Spd accumulation early and transiently during fruit ripening. The ySpdSyn transgenic fruits had a longer shelf life, reduced shriveling and delayed decay symptom development in comparison with the wild‐type (WT) fruits. An increase in shelf life of ySpdSyn transgenic fruits was not facilitated by changes in the rate of water loss or ethylene evolution. Additionally, the expression of several cell wall and membrane degradation‐related genes in ySpdSyn transgenic fruits was not correlated with an extension of shelf life, indicating that the Spd‐mediated increase in fruit shelf life is independent of the above factors. Crop maturity, indicated by the percentage of ripening fruits on the vine, was delayed in a CaMV35S‐ySpdSyn genotype, with fruits accumulating higher levels of the antioxidant lycopene. Notably, whole‐plant senescence in the transgenic plants was also delayed compared with WT plants. Together, these results provide evidence for a role of PAs, particularly Spd, in increasing fruit shelf life, probably by reducing post‐harvest senescence and decay.     

Mechanistic features of Salmonella typhimurium propionate kinase (TdcD): Insights from kinetic and crystallographic studies

Short-chain fatty acids (SCFAs) play a major role in carbon cycle and can be utilized as a source of carbon and energy by bacteria. Salmonella typhimurium propionate kinase (StTdcD) catalyzes reversible transfer of the γ-phosphate of ATP to propionate during l-threonine degradation to propionate. Kinetic analysis revealed that StTdcD possesses broad ligand specificity and could be activated by various SCFAs (propionate > acetate ≈ butyrate), nucleotides (ATP ≈ GTP > CTP ≈ TTP; dATP > dGTP > dCTP) and metal ions (Mg^2 + ≈ Mn^2 + > Co^2 +). Inhibition of StTdcD by tricarboxylic acid (TCA) cycle intermediates such as citrate, succinate, α-ketoglutarate and malate suggests that the enzyme could be under plausible feedback regulation. Crystal structures of StTdcD bound to PO₄ (phosphate), AMP, ATP, Ap4 (adenosine tetraphosphate), GMP, GDP, GTP, CMP and CTP revealed that binding of nucleotide mainly involves hydrophobic interactions with the base moiety and could account for the broad biochemical specificity observed between the enzyme and nucleotides. Modeling and site-directed mutagenesis studies suggest Ala88 to be an important residue involved in determining the rate of catalysis with SCFA substrates. Molecular dynamics simulations on monomeric and dimeric forms of StTdcD revealed plausible open and closed states, and also suggested role for dimerization in stabilizing segment 235–290 involved in interfacial interactions and ligand binding. Observation of an ethylene glycol molecule bound sufficiently close to the γ-phosphate in StTdcD complexes with triphosphate nucleotides supports direct in-line phosphoryl transfer.     

Identification of a hypothetical membrane protein interactor of ribosomal phosphoprotein P0

The ribosomal phosphoprotein P0 of the human malarial parasitePlasmodium falciparum (PfP0) has been identified as a protective surface protein. InDrosophila, P0 protein functions in the nucleus. The ribosomal function of P0 is mediated at the stalk of the large ribosomal subunit at the GTPase centre, where the elongation factor eEF2 binds. The multiple roles of the P0 protein presumably occur through interactions with other proteins. To identify such interacting protein domains, a yeast two-hybrid screen was carried out. Out of a set of sixty clones isolated, twelve clones that interacted strongly with both PfP0 and theSaccharomyces cerevisiae P0 (ScP0) protein were analysed. These belonged to three broad classes: namely (i) ribosomal proteins; (ii) proteins involved in nucleotide binding; and (iii) hypothetical integral membrane proteins. One of the strongest interactors (clone 67B) mapped to the gene YFL034W which codes for a hypothetical integral membrane protein, and is conserved amongst several eukaryotic organisms. The insert of clone 67B was expressed as a recombinant protein, and immunoprecipitaion (IP) reaction with anti-P0 antibodies pulled down this protein along with PfP0 as well as ScP0 protein. Using deletion constructions, the domain of ScP0, which interacted with clone 67B, was mapped to 60–148 amino acids. It is envisaged that the surface localization of P0 protein may be mediated through interactions with putative YFL034W-like proteins inP. falciparum     

Crystal structure of Escherichia coli diaminopropionate ammonia-lyase reveals mechanism of enzyme activation and catalysis

Pyridoxal 5′-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of d- and l-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 Å resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with dl-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp¹²⁰ and Lys⁷⁷ is suggested.     

Mechanism of interaction of O-amino-D-serine with sheep liver serine hydroxymethyltransferase

The mechanism of interaction of O-amino-D-serine (OADS) with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) was established by measuring changes in the enzyme activity, absorption spectra, circular dichroism (CD) spectra, and stopped-flow spectrophotometry. OADS was a reversible noncompetitive inhibitor (Ki = 1.8 microM) when serine was the varied substrate. The first step in the interaction of OADS with the enzyme was the disruption of enzyme-Schiff base, characterized by the rapid disappearance of absorbance at 425 nm (6.5 X 10(3) M-1 s-1) and CD intensity at 430 nm. Concomitantly, there was a rapid increase in absorbance and CD intensity at 390 nm. The spectral properties of this intermediate enabled its identification as pyridoxal 5'-phosphate (PLP). These changes were followed by a slow unimolecular step (2 X 10(-3) s-1) leading to the formation of PLP-OADS oxime, which was confirmed by its absorbance and fluorescence spectra and retention time on high-performance liquid chromatography. The PLP-OADS oxime was displaced from the enzyme by the addition of PLP as evidenced by the restoration of complete enzyme activity as well as by the spectral properties. The unique feature of the mechanism proposed for the interaction of OADS with sheep liver SHMT was the formation of PLP as an intermediate.     

Partial amino acid sequence of sheep liver serine hydroxymethyltransferase and comparison of peptide maps of the enzyme from human, ox livers and Escherichia coli.

A comparison of the tryptic peptide maps of serine hydroxymethyltransferase from sheep, human, ox livers and Escherichia coli revealed that the mammalian enzymes were similar, while the bacterial enzyme exhibited differences in the primary structure. N-terminus of the reduced carboxymethylated sheep liver enzyme was acetylated. Serine hydroxymethyltransferase was hydrolyzed with trypsin and fragments of peptides were separated by high performance liquid chromatography using a combination of gel permeation, reverse phase and ion-pair chromatography. The peptides were sequenced manually using the 4-N,N'-dimethyl aminoazobenzene-4'-isothiocyanate/phenyl isothiocyanate double coupling method. The tryptic peptides with 80% homology or above were aligned on the rabbit liver enzyme sequence.     

Temperature and thiol-induced desensitization of a Ca2+-sensitive cyclic-3',5'-nucleotide phosphodiesterase from sheep lung

Ca2+-sensitivity of sheep lung cyclic-3',5'-nucleotide phosphodiesterase is provided by endogenous tightly bound calmodulin. The calcium sensitivity of a highly purified enzyme was desensitized by increasing the assay temperature. It could also be desensitized to Ca2+-activation by thiols such as dithiothreitol. The thiol-induced desensitization could be partially reversed by dialysis and almost completely reversed by dilution. The results presented in this paper indicate that thiols are possibly involved in the interaction of calmodulin with cyclic-3',5'-nucleotide phosphodiesterase. This is the first report on temperature and thiol-induced desensitization of Ca2+-sensitivity of a cyclic-3',5'-nucleotide phosphodiesterase.     

Crystal structures of open and closed forms of d-serine deaminase from Salmonella typhimurium - implications on substrate specificity and catalysis

Metabolism of D-amino acids is of considerable interest due to their key importance in cell structure and function. Salmonella typhimuriumd-serine deaminase (StDSD) is a pyridoxal 5' phosphate (PLP) dependent enzyme that catalyses degradation of D-Ser to pyruvate and ammonia. The first crystal structure of d-serine deaminase described here reveals a typical Foldtype II or tryptophan synthase β subunit fold of PLP-dependent enzymes. Although holoenzyme was used for crystallization of both wild-type StDSD (WtDSD) and selenomethionine labelled StDSD (SeMetDSD), significant electron density was not observed for the cofactor, indicating that the enzyme has a low affinity for the cofactor under crystallization conditions. Interestingly, unexpected conformational differences were observed between the two structures. The WtDSD was in an open conformation while SeMetDSD, crystallized in the presence of isoserine, was in a closed conformation suggesting that the enzyme is likely to undergo conformational changes upon binding of substrate as observed in other Foldtype II PLP-dependent enzymes. Electron density corresponding to a plausible sodium ion was found near the active site of the closed but not in the open state of the enzyme. Examination of the active site and substrate modelling suggests that Thr166 may be involved in abstraction of proton from the Cα atom of the substrate. Apart from the physiological reaction, StDSD catalyses α, β elimination of D-Thr, D-Allothr and L-Ser to the corresponding α-keto acids and ammonia. The structure of StDSD provides a molecular framework necessary for understanding differences in the rate of reaction with these substrates.