Catalytic Signature Research Articles

The Arabidopsis gene PEPINO/PASTICCINO2 is required for proliferation control of meristematic and non-meristematic cells and encodes a putative anti-phosphatase.

Growth in organisms requires that cell division versus differentiation are precisely controlled and that cells, once differentiated, do not resume proliferation activity. Mutants of the Arabidopsis gene PEPINO/PASTICCINO2 develop abnormal shoot phenotypes from slow tumour-like cell proliferation. Abnormal proliferation is enhanced in a mutant background, which elevates endogenous cytokinin levels as seen in double mutants of pepino and altered meristem primordia1. The enlarged shoot apical meristem produces supernumerary abnormal leaves. However, comparison of expression patterns of Cyclin1At and the homeobox gene KNAT2 indicates that PEPINO ( PEP) is involved in cell cycle regulation of meristematic and non-meristematic cells. PEP encodes a putative anti-phosphatase, a representative of an enigmatic class of proteins, which might participate in proliferation processes. In mammals at least two proteins, Sbf1 and STYX, have been predicted to represent functional anti-phosphatases controlling cell proliferation. However, the in vivo function of this class of molecules is not known since no loss-of-function mutants have been reported to date. Anti-phosphatases display a natural change of the essential cysteine by glycine in the phosphatase catalytic signature motif HCxxGxxR(S/T). The remaining motif harbours highly conserved amino acid residues and is thought to constitute a pocket that enables such proteins to bind but not catalyse phosphorylated residues. One of the sequenced mutations in PEP indicates that the integrity of this anti-phosphatase signature motif is essential for function. The analysis of pepino, the first known loss-of-function mutant of an anti-phosphatase in a multicellular organism, proves the biological significance of these molecules.

A catalytic consensus motif for D-mannitol 2-dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens.

Lys-295, Asn-300 and His-303 of D-mannitol 2-dehydrogenase from Pseudomonas fluorescens were mutated individually into alanine (K295A, N300A and H303A respectively). Purified mutants displayed catalytic efficiencies for NAD(+)-dependent oxidation of D-mannitol 300-fold (H303A), 1000-fold (N300A) and approx. 400000-fold (K295A) below the wild-type level. Comparison of primary kinetic isotope effects on kinetic parameters for D-fructose reduction by wild-type and mutants at pH 10.0 demonstrate that Asn-300 has an auxiliary role in stabilization of the transition state of hydride transfer, and His-303 contributes to substrate positioning. The large solvent isotope effect of 11+/-1 on k (cat) for mannitol oxidation by K295A at pH((2)H) 10.5 suggests a role for Lys-295 in general base enzymic catalysis. Positional conservation of Lys-295, Asn-300 and His-303 across a family of polyol-specific long-chain dehydrogenases suggests a unique catalytic signature: Lys-Xaa(4)-Asn-Xaa(2)-His (where 'Xaa' denotes 'any amino acid').

M13 endopeptidases: New conserved motifs correlated with structure, and simultaneous phylogenetic occurrence of PHEX and the bony fish

M13 endopeptidase alignments have focused mainly on mammalian sequences and on the active site region defining the catalytic sequence signatures. Aligning all available M13 from bacteria to human on a full-length basis, we have performed a sequence analysis. This enabled us to highlight the origin and function of the M13 PHEX subtype family endopeptidase (phosphate regulating gene with homologies to endopeptidases on the X chromosome). New evolutionary conserved regions in both prokaryotes and eukaryotes have been detected and eukaryotic-specific regions clearly delineated. Using the recently solved neprilysin structure, we have observed that all new motifs, except one, localize in the spatial vicinity of the previously reported catalytic signatures. Interestingly, a highly hydrophobic pocket containing three newly reported motifs is centered by the C-terminal tryptophan residue. Extensive M13 searches in complete and in progress higher eukaryotic genomes have lead to the identification of Danio rerio as the simplest organism having PHEX. Finally, the human PHEX substrate, the parathyroid hormone-related peptide, PTHrP(107-139), is absent in bony fish: this suggests the existence of further PHEX substrates common to both bony fishes and higher vertebrates.

Exploring the active site of yeast xylose reductase by site-directed mutagenesis of sequence motifs characteristic of two dehydrogenase/reductase family types

Starting from a common tyrosine, yeast xylose reductases (XRs) contain two conserved sequence motifs corresponding to the catalytic signatures of single-domain reductases/epimerases/dehydrogenases (Tyr n -(X) 3-Lys n+4 ) and aldo/keto reductases (AKRs) (Tyr n -(X) 28-Lys n+29 ). Tyr 51, Lys 55 and Lys 80 of XR from Candida tenuis were replaced by site-directed mutagenesis. The purified Tyr 51→ Phe and Lys 80→Ala mutants showed turnover numbers and catalytic efficiencies for NADH-dependent reduction of D-xylose between 2500- and 5000-fold below wild-type levels, suggesting a catalytic role of both residues. Replacing Lys 55 by Asn, a substitution found in other AKRs, did not detectably affect binding of coenzymes, and enzymatic catalysis to carbonyl/alcohol interconversion. The contribution of Tyr 51 to rate enhancement of aldehyde reduction conforms with expectations for the general acid catalyst of the enzymatic reaction.

2.9 Å crystal structure of ligand‐free tryptophanyl‐tRNA synthetase: Domain movements fragment the adenine nucleotide binding site

The crystal structure of ligand-free tryptophanyl-tRNA synthetase (TrpRS) was solved at 2.9 A using a combination of molecular replacement and maximum-entropy map/phase improvement. The dimeric structure (R = 23.7, Rfree = 26.2) is asymmetric, unlike that of the TrpRS tryptophanyl-5'AMP complex (TAM; Doublié S, Bricogne G, Gilmore CJ, Carter CW Jr, 1995, Structure 3:17-31). In agreement with small-angle solution X-ray scattering experiments, unliganded TrpRS has a conformation in which both monomers open, leaving only the tryptophan-binding regions of their active sites intact. The amino terminal alphaA-helix, TIGN, and KMSKS signature sequences, and the distal helical domain rotate as a single rigid body away from the dinucleotide-binding fold domain, opening the AMP binding site, seen in the TAM complex, into two halves. Comparison of side-chain packing in ligand-free TrpRS and the TAM complex, using identification of nonpolar nuclei (Ilyin VA, 1994, Protein Eng 7:1189-1195), shows that significant repacking occurs between three relatively stable core regions, one of which acts as a bearing between the other two. These domain rearrangements provide a new structural paradigm that is consistent in detail with the "induced-fit" mechanism proposed for TyrRS by Fersht et al. (Fersht AR, Knill-Jones JW, Beduelle H, Winter G, 1988, Biochemistry 27:1581-1587). Coupling of ATP binding determinants associated with the two catalytic signature sequences to the helical domain containing the presumptive anticodon-binding site provides a mechanism to coordinate active-site chemistry with relocation of the major tRNA binding determinants.

Reduction of SO4=Ions in Sulfated Zirconia Catalysts

Upon reducing sulfated zirconia (SZ) catalysts with hydrogen, roughly 50% of the sulfate groups are reduced to sulfur dioxide which is detected mass spectrometrically; the other 50% are reduced to S=ions that are retained at the surface. The measured ratio H/S of consumed H atoms to S atoms originally present as SO4=ions is thus H/S=5. In the presence of platinum, either deposited on the SZ, or as Pt/NaY in a physical mixture with SZ, or downstream of SZ in a layered bed arrangement, the SO2is reduced further to H2S, which is detected chemically and by MS. In this case H/S=8. The TPR peak position is shifted to lower temperature in PtSZ and in physical mixtures of SZ and Pt/NaY. Reduction of sulfate groups lowers the Brønsted acidity of the catalysts, as indicated by theintensityof the IR bands of adsorbed ammonia. Brønsted acidity is almost totally eliminated by hydrogen reduction at 400°C. The position of the band of ammonia on Lewis sites is not significantly affected by sulfate reduction, but the temperature at which ammonia is desorbed from these sites is lowered. The H2S that is formed in the presence of Pt partially poisons the Pt particles; their catalytic signature in the isotope exchange of cyclopentane with D2indicates that large Pt ensembles are blocked by adsorbed S atoms even after reduction up to 350°C, but that isolated Pt atoms are still acting as active sites. After complete reduction of the SO4=groups, Pt in PtSZ loses its catalytic activity for the isotope exchange reaction.