ABSTRACT
The cDNA for pre-pro-Concanavalin A (pre-pro-ConA) was cloned into the cytoplasmic expression vector pKK233-2 to give rise to pCONEXP2 which was used to express the lectin precursor. Pre-pro-ConA is stable and is not transposed and ligated to form the mature protein. No signal peptide removal is observed. The solubility of pre-pro-ConA could not be increased by guanidine hydrochloride denaturation/dilution treatment.
Subject(s)
Concanavalin A/chemistry , Fabaceae/chemistry , Protein Precursors/chemistry , Blotting, Western , Cloning, Molecular , Concanavalin A/genetics , Plant Lectins , Protein Precursors/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , SolubilityABSTRACT
DNA coding for the ferredoxin-dependent glutamate synthase (EC 1.4.7.1) of spinach chloroplasts has been cloned and sequenced. It consists of 5015 bp and starts with the codon for the N-terminal cysteine of the mature protein. Ferredoxin-dependent glutamate synthase is one of the key enzymes in the early stages of ammonia assimilation in plants, algae and cyanobacteria. In addition to the ferredoxin-dependent enzyme, there are two other forms of glutamate synthase, one of which uses NADH as the electron donor and a second that uses NADPH. Although all three forms catalyze the reductive transamidation of the amido nitrogen from glutamine to 2-oxoglutarate to form two molecules of glutamate, ferredoxin-dependent glutamate synthases differ from the NADH and NADPH-dependent forms in subunit composition and amino acid sequence. The recent availability of sequence data for glutamate synthases from spinach and from two archael species has produced a clearer and more detailed picture of the evolution of this key enzyme in nitrogen metabolism and the origins of the two subunit/domain structure of the enzyme.
Subject(s)
Evolution, Molecular , Glutamate Synthase/genetics , Amino Acid Oxidoreductases/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Cloning, Molecular , Molecular Sequence Data , NADP/chemistry , Plant Proteins/genetics , Sequence Alignment , Sequence Analysis, DNA , Spinacia oleracea/enzymology , Substrate SpecificityABSTRACT
The nucleotide sequence of a 1634 bp DNA fragment from the photosynthetic purple sulfur bacterium Allochromatium vinosum contains one complete and two partial open reading frames. Sequence comparisons to genes from other organisms suggest that this A. vinosum DNA fragment contains, starting from the 5' end, the following: (1) 234 bp at the 3' end of the A. vinosum purH gene, coding for 78 amino acids at the C-terminus of the bi-functional 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide formyltransferase/IMP cyclohydrolase (EC 2.1.2.3), an enzyme involved in de novo purine biosynthesis; (2) 777 bp of the A. vinosum lpxA gene, coding for all 259 amino acids of the UDP-N-acetylglucosamine-O-acyltransferase, an enzyme involved in lipid A biosynthesis; and (3) 567 bp at the 5' end of the A. vinosum purD gene, coding for 189 amino acids at the N-terminus of 5'-phosphoribosyl glycinamide synthetase (EC 6.3.4.13), a second enzyme involved in de novo purine biosynthesis. The presence of a gene coding for an enzyme involved in lipid A biosynthesis between two genes coding for enzymes of the de novo purine biosynthesis pathway represents a unique arrangement of these genes.
