<p>Acetolactate synthase (ALS) and acetohydroxyacid
synthase (AHAS) are two thiamin diphosphate (ThDP)-dependent enzymes that
catalyze the formation of acetolactate from two molecules of pyruvate. In addition
to acetolactate, AHAS can catalyze the formation of acetohydroxybutyrate from
pyruvate and α-ketobutyrate. When formed by AHAS, these compounds are important
precursors to the essential amino acids valine and isoleucine. Conversely, ALS
forms acetolactate as a precursor to 2,3‑butanediol, a product formed in an
alternative pathway to mixed acid fermentation.</p>
<p>While these enzymes catalyze the same reaction,
they have been found to be quite different. Such differences include:
biological function, pH optimum, cofactor requirements, reaction kinetics and
quaternary structure. Importantly, AHAS has been identified as the target of
the widely-used sulfonylurea and imidazolinone herbicides, which has led to
many structural and kinetic studies on AHAS enzymes from plants, bacteria, and
fungi. ALS, on the other hand, has only been identified in bacteria, and has
largely not seen such extensive characterization. Finally, although some
bacteria contain both enzymes, they have never been studied in detail from the
same organism. </p>
<p>Here, the ALS and AHAS enzymes from <i>Klebsiella pneumoniae</i> were studied using
steady-state kinetic analyses, X-ray crystallography, site-directed and site‑saturation
mutagenesis, and cell growth complementation assays to i) compare the kinetic
parameters of each enzyme, ii) compare the active sites to probe their
differences in substrate profile and iii) test the ability of ALS to function
in place of AHAS <i>in vivo</i>.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/8263847 |
| Date | 16 October 2019 |
| Creators | Alexander Jon Latta (6831542) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/Structural_and_Kinetic_Comparison_of_Acetolactate_Synthase_and_Acetohydroxyacid_Synthase_from_i_Klebsielle_pneumoniae_i_/8263847 |
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