The first step of aminoacylation at the atomic level in histidyl-tRNA synthetase

The crystal structure of an enzyme–substrate complex with histidyl-tRNA synthetase from Escherichia coli, ATP, and the amino acid analog histidinol is described and compared with the previously obtained enzyme–product complex with histidyl-adenylate. An active site arginine, Arg-259, unique to all histidyl-tRNA synthetases, plays the role of the catalytic magnesium ion seen in seryl-tRNA synthetase. When Arg-259 is substituted with histidine, the apparent second order rate constant (kcat/Km) for the pyrophosphate exchange reaction and the aminoacylation reaction decreases 1,000-fold and 500-fold, respectively. Crystals soaked with MnCl2 reveal the existence of two metal binding sites between β- and γ-phosphates; these sites appear to stabilize the conformation of the pyrophosphate. The use of both conserved metal ions and arginine in phosphoryl transfer provides evidence of significant early functional divergence of class II aminoacyl-tRNA synthetases.

An Embryo-Defective Mutant of Arabidopsis Disrupted in the Final Step of Biotin Synthesis

Auxotrophic mutants have played an important role in the genetic dissection of biosynthetic pathways in microorganisms. Equivalent mutants have been more difficult to identify in plants. The bio1 auxotroph of Arabidopsis thaliana was shown previously to be defective in the synthesis of the biotin precursor 7,8-diaminopelargonic acid. A second biotin auxotroph of A. thaliana has now been identified. Arrested embryos from this bio2 mutant are defective in the final step of biotin synthesis, the conversion of dethiobiotin to biotin. This enzymatic reaction, catalyzed by the bioB product (biotin synthase) in Escherichia coli, has been studied extensively in plants and bacteria because it involves the unusual addition of sulfur to form a thiophene ring. Three lines of evidence indicate that bio2 is defective in biotin synthase production: mutant embryos are rescued by biotin but not dethiobiotin, the mutant allele maps to the same chromosomal location as the cloned biotin synthase gene, and gel-blot hybridizations and polymerase chain reaction amplifications revealed that homozygous mutant plants contain a deletion spanning the entire BIO2-coding region. Here we describe how the isolation and characterization of this null allele have provided valuable insights into biotin synthesis, auxotrophy, and gene redundancy in plants.

Cloning, baculovirus expression, and characterization of a second mouse prolyl 4-hydroxylase alpha-subunit isoform: formation of an alpha 2 beta 2 tetramer with the protein disulfide-isomerase/beta subunit.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer, the beta subunit of which is a highly unusual multifunctional polypeptide, being identical to protein disulfide-isomerase (EC 5.3.4.1). We report here the cloning of a second mouse alpha subunit isoform, termed the alpha (II) subunit. This polypeptide consists of 518 aa and a signal peptide of 19 aa. The processed polypeptide is one residue longer than the mouse alpha (I) subunit (the previously known type), the cloning of which is also reported here. The overall amino acid sequence identity between the mouse alpha (II) and alpha (I) subunits is 63%. The mRNA for the alpha (II) subunit was found to be expressed in a variety of mouse tissues. When the alpha (II) subunit was expressed together with the human protein disulfide-isomerase/beta subunit in insect cells by baculovirus vectors, an active prolyl 4-hydroxylase was formed, and this protein appeared to be an alpha (II) 2 beta 2 tetramer. The activity of this enzyme was very similar to that of the human alpha (I) 2 beta 2 tetramer, and most of its catalytic properties were also highly similar, but it differed distinctly from the latter in that it was inhibited by poly(L-proline) only at very high concentrations. This property may explain why the type II enzyme was not recognized earlier, as an early step in the standard purification procedure for prolyl 4-hydroxylase is affinity chromatography on a poly(L-proline) column.