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Acetohydroxyacid synthase (AHAS) is one of the key enzymes of the biosynthesis of branched-chain amino acids, it is also an effective target for the screening of herbicides and antibiotics. In this study we present a method for preparing Escherichia coli AHAS I holoenzyme (EcAHAS I) with exceptional stability, which provides a solid ground for us to re-investigate the in vitro catalytic properties of the protein. The results show EcAHAS I synthesized in this way exhibits similar function to Bacillus subtilis acetolactate synthase in its catalysis with pyruvate and 2-ketobutyrate (2-KB) as dual-substrate, producing four 2-hydroxy-3-ketoacids including (S)-2-acetolactate, (S)-2-aceto-2-hydroxybutyrate, (S)-2-propionyllactate, and (S)-2-propionyl-2-hydroxybutyrate. Quantification of the reaction indicates that the two substrates almost totally consume, and compound (S)-2-aceto-2- hydroxybutyrate forms in the highest yield among the four major products. Moreover, the protein also condenses two molecules of 2-KB to furnish (S)-2-propionyl-2-hydroxybutyrate. Further exploration manifests that EcAHAS I ligates pyruvate/2-KB and nitrosobenzene to generate two arylhydroxamic acids N -hydroxy- N -phenylacetamide and N -hydroxy- N -phenyl- propionamide. These findings enhance our comprehension of the catalytic characteristics of EcAHAS I. Furthermore, the application of this enzyme as a catalyst in construction of C –N bonds displays promising potential. [Display omitted] • The in vitro activity of AHAS I from E. coli (EcAHAS) was re-investigated. • EcAHAS condenses pyruvate or 2-ketobutyrate (2-KB) to furnish (S)-AL or (S)-PHB. • It condenses pyruvate and 2-KB to produce (S)-AL, (S)-AHB, (S)-PL, and (S)-PHB. • It ligates pyruvate and benzylaldehyde to give (R)-PAC. • It ligates pyruvate/2-KB and nitrosobenzene to yield arylhydroxamic acids. [ABSTRACT FROM AUTHOR]