Structure and expression of a cyanobacterial ilvC gene encoding acetohydroxyacid isomeroreductase

Abstract

Acetohydroxyacid isomeroreductase (AHAIR) is the shared second enzyme in the biosynthetic pathways leading to isoleucine and valine. AHAIR is encoded by the ilvC gene in bacteria. A 1,544-bp fragment of genomic DNA containing the ilvC gene was cloned from the cyanobacterium Synechocystis sp. strain PCC 6803, and the complete nucleotide sequence was determined. The identity of the gene was established by comparison of the nucleotide and derived peptide sequences with those of other ilvC genes. The highest degree of sequence similarity was found with the ilvC gene from Rhizobium meliloti. The isolated Synechocystis ilvC gene complemented an Escherichia coli ilvC mutant lacking AHAIR activity. The expressed Synechocystis gene encodes a protein that has a molecular mass of 35.7 kDa and that has AHAIR activity in an in vitro assay. Polyclonal antibodies raised against purified Synechocystis AHAIR produced a single band on a Western blot (immunoblot) of a Synechocystis cell extract and detected the protein in an extract of an E. coli ilvC mutant strain that was transformed with a plasmid containing the Synechocystis ilvC gene. The antibody did not react with an extract of an E. coli ilvC mutant strain that was transformed with a control plasmid lacking the Synechocystis ilvC gene or with an extract of an E. coli IlvC+ control strain.

Evidence for two catalytically different magnesium-binding sites in acetohydroxy acid isomeroreductase by site-directed mutagenesis

“…meliloti --------------------------------------------------E --------------------------FLAQ --------------------------FLAQ --------- R. meliloti E.coli between the acetohydroxy acid isomeroreductases from S. oleracea (Dumas et al, 1991), A. thaliana (Curien et al, 1993), Saccharomyces cerevisiae (Petersen & Holmberg, 1986), Neurospora crassa (Sista & Bowman, 1992), Lactobacillus lactis (Godon et al, 1992), Synechocystis sp. (Rieble & Beale, 1992), Rhizobium meliloti (Aguilar & Grasso, 1991), and E. coli (Wek & Hatfield, 1986). They define the conserved domains I-V. A conserved sequence not found in the R. meliloti sequence was underlined.…”

“…To identify essential domains or residues of the enzyme that participate in metal ion cofactor binding, the predicted amino acid sequences of plant acetohydroxy acid isomer-oreductase have been aligned to the known sequences of corresponding enzymes from fungi (Petersen & Holmberg, 1986;Sista & Bowman, 1992) and bacteria (Blazey & Bums, 1984; Wek & Hatfield, 1986;Aguilar & Grasso, 1991;Godon et al, 1992;Rieble & Beale, 1992) (Figure 2). This sequence comparison indicated five regions of identity that have been designated in this paper as domains I-V. Domain I is similar to the fingerprint region of the NADPH-binding site found in a large number of NADPH-dependent oxidoreductases (Dumas et al, 1991).…”

Regulation by external pH and stationary growth phase of the acetolactate synthase from Synechocystis PCC6803

“…In Synechocystis PCC6803, genes have been identified for an acetohydroxy acid isomeroreductase (Fig. 1) (Rieble and Beale, 1992) and for two potential large subunits (ORFs sll1981 and slr2088 ) and a small subunit (ORF sll0065 ) of ALS/AHAS (Kaneko et al ., 1996). The slr2088 ‐encoded protein shows high homology to the AHAS enzyme of S. platensis (Milano et al ., 1992).…”

Integrated biomarker for type 2 diabetes mellitus and impaired fasting glucose based on metabolomics analysis using ultra‐high performance liquid chromatography quadrupole‐Orbitrap high‐resolution accurate mass spectrometry

“…As the FBG concentration increased in T2DM patients, the level of pyruvic acid was increased, which would lead to an increase in 2‐acetolactate 39 . Then, under the action of various active enzymes, such as acetohydroxy acid isomeroreductase and branched‐chain amino acid transaminase, 2‐acetolactate undergoes a biosynthetic pathway to form valine and isoleucine 40 . The results of this study showed that the FC values of L‐valine and L‐isoleucine were 1.21 and 1.22, which conformed to the above pathway.…”