Cloning and Characterization of Acetohydroxyacid Synthase from Bacillus stearothermophilus

Abstract

Five genes from the ilv-leu operon from Bacillus stearothermophilus have been sequenced. Acetohydroxyacid synthase (AHAS) and its subunits were separately cloned, purified, and characterized. This thermophilic enzyme resembles AHAS III of Escherichia coli, and regulatory subunits of AHAS III complement the catalytic subunit of the AHAS of B. stearothermophilus, suggesting that AHAS III is functionally and evolutionally related to the single AHAS of gram-positive bacteria.The first step common to the biosynthesis of branchedchain amino acids, catalyzed by acetohydroxyacid synthase (AHAS) (EC 4.1.3.18), is the condensation of pyruvate with either 2-ketobutyrate (the precursor of isoleucine) or pyruvate (the precursor of valine) (4, 26). Bacterial AHASs are composed of large (60-kDa) catalytic and small (9-to 18-kDa) regulatory subunits. Isolated catalytic subunits have lower activity than the holoenzymes but are similar to them in their cofactor dependence and specificity towards the two different substrates (10,27,28). The sensitivity of AHAS to feedback inhibition is completely dependent on the small subunit.Many bacteria and archaea apparently contain a single AHAS enzyme. In most gram-positive bacteria, the genes for the first two enzymes in the pathway are located in the same operon (ilvBNC) (5,9,13,15,30), often together with the leu genes (ilvBNC-leuACBD) (17,25,30). The enterobacteria contain three isozymes of AHAS, encoded by distinct and differently regulated operons (3, 4).To investigate the AHAS of Bacillus stearothermophilus (AHAS Bst ), we cloned the genes for this holoenzyme (ilvBN) and its large (ilvB) and small (ilvN) subunits to allow sequencing and overexpression. The screening for these genes was conducted with a genomic cosmid library for B. stearothermophilus ATCC 7954, created by H. Ewis (unpublished data), with a digitonin-labeled 1,100-bp probe that is highly conserved (50 to 75% amino acid identity) among AHASs (7) and only slightly conserved in other thiamine diphosphate (ThDP)-dependent enzymes, such as pyruvate oxidase (30%) and catabolic acetolactate synthase (25%) of Bacillus subtilis. This probe was amplified from the B. stearothermophilus ATCC 12980 genome by using two degenerate oligonucleotide primers: 5Ј(C/T/A)GGNACNGA(T/C)GCNTT(T/C)CA(A/G)GA and 5Ј T(C/G)(C/T)TGCCA(C/T)(T/G)NACCAT.The gene order in the insert of the AHAS-positive cosmid, as determined by coding analysis of its sequence (Fig. 1), seems similar to that of the B. subtilis ilv-leu operon (16, 30). The 5Ј end of ilvB was absent in the cosmid-cloned fragment. This region was added to the clone, as shown in Fig. 1, from a PCR-amplified fragment obtained from the genome of B. stearothermophilus ATCC 7954 by using primers that were identical to the T-box element of the ilv-leu operon from B. subtilis (14) (GGGTGGTACCGCGG) and to a sequenced 3Ј region of ilvB from B. stearothermophilus (GGCGGATTTGC CAATGGTTCGGC).The DNA sequences of the ilv-leu operon of B. stearothermophilus (NCBI accession no. AY083837) and the deduced...

Influence of Allosteric Regulators on Individual Steps in the Reaction Catalyzed by Mycobacterium tuberculosis 2-Hydroxy-3-oxoadipate Synthase

“…) are comparable with those of other carboligases that use 2-ketoacid substrates, such as glyoxylate carboligase and isozymes of acetohydroxyacid synthase from various bacteria (17,25,(27)(28)(29)(30). The rates on the unactivated enzyme are similar to those of M. tuberculosis acetohydroxyacid synthase, whereas the activated rates are comparable with those of E. coli enzymes.…”

The distribution of acetohydroxyacid synthase in soil bacteria

“…Previous studies have utilized oligonucleotide probes in the study of AHAS in archaea (Bowen et al 1997) and bacteria (Porat et al 2004), but these probes were specific for the microorganisms being studied. We have designed primers that appeared to amplify DNA encoding a section of the IlvB conserved domain, regardless of the type of AHAS isozyme.…”

“…Although evidence of isozymes is not extensive, they have been observed in cyanobacteria (Forlani et al 1991). Recently, Porat et al (2004) characterized a thermophilic AHAS enzyme from B. stearothermophilus, which resembles E. coli AHAS III, and is encoded for by the genes ilvB and ilvN. Some cProteobacteria also appear to contain AHAS isozymes, including Vibrio vulnificus (Kim et al 2003) and a recently characterized marine bacterium, Colwellia psychrerythrea (Methe et al 2005).…”

Crystal structures of TM0549 and NE1324—two orthologs of E. coli AHAS isozyme III small regulatory subunit

“…The large catalytic subunits also show very weak activity without their associated regulatory subunits (Weinstock et al 1992;Pang and Duggleby 1999). The E. coli AHAS enzyme can be reconstituted with SSU subunits from other organisms (Porat et al 2004), and the reconstituted enzymes form stable heterotetramers. The properly associated holoenzymes show full catalytic activity, and are also susceptible to valine inhibition.…”