Bernd Laber

The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction.

A stoichiometric complex of human stefin B and carboxymethylated papain has been crystallized in a trigonal crystal form. Data to 2.37 A resolution were collected using the area detector diffractometer FAST. The crystal structure of the complex has been solved by Patterson search techniques using papain as search model. Starting from the structure of chicken cystatin, the stefin structure was elucidated through cycles of model building and crystallographic refinement. The current crystallographic R factor is 0.19. Like cystatin, the stefin molecule consists of a five stranded beta‐sheet wrapped around a five turn alpha‐helix, but with an additional carboxy terminal strand running along the convex side of the sheet. Topological equivalence of stefin and cystatin reveal the previous sequence alignment to be incorrect in part, through deletion of the intermediate helix. The conserved residues form a tripartite wedge, which slots into the papain active site as proposed through consideration of the tertiary structures of the individual components (Bode et al., 1988). The main interactions are provided by the amino terminal ‘trunk’ (occupying the ‘unprimed’ subsites of the enzyme), and by the first hairpin loop, containing the highly conserved QVVAG sequence, with minor contributions from the second hairpin loop. The carboxyl terminus of stefin provides an additional interaction region with respect to cystatin. The interaction is dominated by hydrophobic contacts. Inhibition by the cysteine proteinase inhibitors is fundamentally different to that observed for the serine proteinase inhibitors.

Vitamin B6 biosynthesis: formation of pyridoxine 5′-phosphate from 4-(phosphohydroxy)-l-threonine and 1-deoxy-d-xylulose-5-phosphate by PdxA and PdxJ protein

In Escherichia coli the coenzyme pyridoxal 5′‐phosphate (PLP) is synthesised de novo by a pathway that is thought to involve the condensation of 4‐(phosphohydroxy)‐l‐threonine and 1‐deoxy‐d‐xylulose, catalysed by the enzymes PdxA and PdxJ, to form either pyridoxine (vitamin B6) or pyridoxine 5′‐phosphate (PNP). Here we show that incubation of PdxJ with PdxA, 4‐(phosphohydroxy)‐l‐threonine, NAD and 1‐deoxy‐d‐xylulose‐5‐phosphate, but not 1‐deoxy‐d‐xylulose, results in the formation of PNP. The PNP formed was characterised by (i) cochromatography with an authentic standard, (ii) conversion to pyridoxine by alkaline phosphatase treatment, and (iii) UV and fluorescence spectroscopy. Furthermore, when [2‐14C]1‐deoxy‐d‐xylulose‐5‐phosphate was used as a substrate, the radioactivity was incorporated into PNP. These results clarify the previously unknown role of PdxJ in the de novo PLP biosynthetic pathway. The sugar used as substrate by PdxJ is 1‐deoxy‐d‐xylulose‐5‐phosphate rather than the previously assumed 1‐deoxy‐d‐xylulose. The first vitamin B6 vitamer synthesised is PNP, and not pyridoxine.

Determinants of enzymatic specificity in the Cys-Met-metabolism PLP-dependent enzyme family: Crystal structure of cystathionine γ-lyase from yeast and intrafamiliar structure comparison

Abstract The crystal structure of cystathionine γ-lyase (CGL) from yeast has been solved by molecular replacement at a resolution of 2.6 å. The molecule consists of 393 amino acid residues and one PLP moiety and is arranged in the crystal as a tetramer with D2 symmetry as in other related enzymes of the CysMetmetabolism PLP-dependent family like cystathionine β-lyase (CBL). A structure comparison with other family members revealed surprising insights into the tuning of enzymatic specificity between the different family members. CGLs from yeast or human are virtually identical at their active sites to cystathionine γ-synthase (CGS) from E. coli. Both CGLs and bacterial CGSs exhibit γ-synthase and γ-lyase activities depending on their position in the metabolic pathway and the available substrates. This group of enzymes has a glutamate (E333 in yeast CGL) which binds to the distal group of cystathionine (CTT) or the amino group of cysteine. Plant CGSs use homoserine phosphate instead of O-succinyl-homoserine as one substrate. This is reflected by a partially different active site structure in plant CGSs. In CGL and CBL the pseudosymmetric substrate must dock at the active site in different orientations, with S in γ-position (CBL) or in δ-position (CGL). The conserved glutamate steers the substrate as seen in other CGLs. In CBLs this position is occupied by either tyrosine or hydrophobic residues directing binding of CTT such that S is in the in γ-position. In methionine γ-lyase a hydrophic patch operates as recognition site for the methyl group of the methionine substrate.

Repression of Acetolactate Synthase Activity through Antisense Inhibition (Molecular and Biochemical Analysis of Transgenic Potato (Solanum tuberosum L. cv Desiree) Plants)

Acetolactate synthase (ALS), the first enzyme in the biosynthetic pathway of leucine, valine, and isoleucine, is the biochemical target of different herbicides. To investigate the effects of repression of ALS activity through antisense gene expression we cloned an ALS gene from potato (Solanum tuberosum L. cv Desiree), constructed a chimeric antisense gene under control of the cauliflower mosaic virus 35S promoter, and created transgenic potato plants through Agrobacterium tumefaciens-mediated gene transfer. Two regenerants revealed severe growth retardation and strong phenotypical effects resembling those caused by ALS-inhibiting herbicides. Antisense gene expression decreased the steady-state level of ALS mRNA in these plants and induced a corresponding decrease in ALS activity of up to 85%. This reduction was sufficient to generate plants almost inviable without amino acid supplementation. In both ALS antisense and herbicide-treated plants, we could exclude accumulation of 2-oxobutyrate and/or 2-aminobutyrate as the reason for the observed deleterious effects, but we detected elevated levels of free amino acids and imbalances in their relative proportions. Thus, antisense inhibition of ALS generated an in vivo model of herbicide action. Furthermore, expression of antisense RNA to the enzyme of interest provides a general method for validation of potential herbicide targets.

The cysteine proteinase inhibitor chicken cystatin is a phosphoprotein

Peptide maps obtained by reversed‐phase HPLC of tryptic digests of isoelectric form 1 (pI=6.5) and 2 (pI=5.6) of chicken egg white cystatin revealed that the difference was located only in a single peptide (residues Ser‐74—Lys‐91). Ser‐80 of cystatin 2 was subsequently identified as being modified by phosphorylation. Moreover, alkaline phosphatase treatment of a mixture of native cystatin forms 1 and 2 was shown by ion‐exchange chromatography to cause the disappearance of isoelectric form 2 with a concomitant increase in form 1. Thus, the existence of two isoelectric forms of chicken cystatin is due to the phosphorylated form 2 and non‐phosphorylated form 1.

Purification and Properties of Cystathionine [gamma]-Synthase from Wheat (Triticum aestivum L.).

Cysthathionine [gamma]-synthase (CS), an enzyme involved in methionine biosynthesis, was purified from an acetone powder prepared from wheat (Triticum aestivum L.). After several chromatographic steps and radiolabeling of the partially purified enzyme with sodium cyanoboro[3H]hydride, a single polypeptide with a molecular weight of 34,500 was isolated by sodium dodecyl sulfate-high performance electrophoresis chromatography. Since the molecular weight of the native enzyme was 155,000, CS apparently consists of four identical subunits. The pyridoxal 5[prime]-phosphate-dependent forward reaction has a pH optimum of 7.5 and follows a hybrid ping-pong mechanism with Km values of 3.6 mM and 0.5 mM for L-homoserine phosphate and L-cysteine, respectively. L-Cysteine methyl ester, thioglycolate methyl ester, and sodium sulfide were also utilized as thiol substrates. The latter observation suggests that CS and phosphohomoserine sulfhydrase might be a single enzyme. CS does not seem to be a regulatory enzyme but was irreversibly inhibited by DL-propargylglycine (Ki = 45 [mu]M, Kinact = 0.16 min-1). Furthermore, the homoserine phosphate analogs 4-(phosphonomethyl)-pyridine-2-carboxylic acid, Z-3-(2-phosphonoethen-1-yl)pyridine-2-carboxylic acid, and DL-E-2-amino-5-phosphono-3-pentenoic acid acted as reversible competitive inhibitors with Ki values of 45, 40, and 1.1 [mu]M, respectively.

Structure and function of threonine synthase from yeast

Threonine synthase catalyzes the final step of threonine biosynthesis, the pyridoxal 5′-phosphate (PLP)-dependent conversion ofO-phosphohomoserine into threonine and inorganic phosphate. Threonine is an essential nutrient for mammals, and its biosynthetic machinery is restricted to bacteria, plants, and fungi; therefore, threonine synthase represents an interesting pharmaceutical target. The crystal structure of threonine synthase from Saccharomyces cerevisiae has been solved at 2.7 Å resolution using multiwavelength anomalous diffraction. The structure reveals a monomer as active unit, which is subdivided into three distinct domains: a small N-terminal domain, a PLP-binding domain that covalently anchors the cofactor and a so-called large domain, which contains the main of the protein body. All three domains show the typical open α/β architecture. The cofactor is bound at the interface of all three domains, buried deeply within a wide canyon that penetrates the whole molecule. Based on structural alignments with related enzymes, an enzyme-substrate complex was modeled into the active site of yeast threonine synthase, which revealed essentials for substrate binding and catalysis. Furthermore, the comparison with related enzymes of the β-family of PLP-dependent enzymes indicated structural determinants of the oligomeric state and thus rationalized for the first time how a PLP enzyme acts in monomeric form.

CLONING, PURIFICATION AND CHARACTERISATION OF CYSTATHIONINE GAMMA -SYNTHASE FROM NICOTIANA TABACUM

Abstract Cystathionine γ-synthase, the enzyme catalysing the first reaction specific for methionine biosynthesis, has been cloned from Nicotiana tabacum, overexpressed in Escherichia coli and purified to homogeneity. The recombinant cystathionine γ-synthase catalyses the pyridoxal 5′-phosphate dependent formation of L-cystathionine from L-homoserine phosphate and L-cysteine with apparent Km-values of 7.1 ± 3.1mM and of 0.23 ± 0.07 mM, respectively. The enzyme was irreversibly inhibited by DL-propargylglycine (K i = 18 ΜM, k inact = 0.56 min−1), while the homoserine phosphate analogues 3-(phosphonomethyl)pyridine-2-carboxylic acid, 4-(phosphonomethyl)pyridine-2-carboxylic acid, Z-3-(2-phosphonoethen-1-yl)pyridine-2-carboxylic acid, and DL-E-2-amino-5-phosphono-3-pentenoic acid acted as reversible competitive inhibitors with K i values of 0.20, 0.30, 0.45, and 0.027mM, respectively. In combination these results suggest a ping-pong mechanism for the cystathionine γ-synthase reaction, with homoserine phosphate binding to the enzyme first. Large single crystals of cystathionine γ-synthase diffracting to beyond 2.7 Å resolution were obtained by the sitting drop vapour diffusion method. The crystals belong to the orthorhombic space group P212121 with unit cell constants a = 120.0 Å, b = 129.5 Å, c = 309.8 Å, corresponding to two tetramers per asymmetric unit.

Inhibition of phenylalanine ammonia-lyase in vitro and in vivo by (1-amino-2-phenylethyl)phosphonic acid, the phosphonic analogue of phenylalanine

Abstract The phosphonic analogue of ʟ-phenylalanine, (R)-(1-amino-2-phenylethyl)phosphonic acid (APEP), inhibits buckwheat phenylalanine ammonia-lyase (PAL) competitively with a Ki value of 1.5 μM. The Ki value for the (S)-enantiomer is 11.6 μM. The corresponding values for the enantiomers of the phosphonous analogue are 35 and 205 μm , respectively. APEP inhibits the light-induced synthesis of anthocyanin in hypocotyls of etiolated buckwheat seedlings and causes a specific increase in the endogenous phenylalanine concentration in buckwheat hypocotyls as well as other plant tissues. Kohlrabi seedlings develop normally in the presence of APEP, while their anthocyanin content is greatly reduced. These results indicate that APEP inhibits PAL in vivo.

Cloning, purification, and crystallization of Escherichia coli cystathionine β-lyase

The metC gene coding for cystathionine β‐lyase of Escherichia coli has been cloned and used to construct an overproducing E. coli strain. An efficient purification scheme has been developed and the purified enzyme has been crystallized by the hanging drop vapour diffusion method using either ammonium sulfate or polyethyleneglycol 400 as precipitating agent. The crystals belong to the orthorombic space group C2221. Their unit cell parameters are . Consideration of the possible values of V M accounts for the presence of one dimer per asymmetric unit. The crystals are suitable for X‐ray analysis and a complete native date set to 1.83 Å resolution has been collected using synchrotron radiation.