alpha-Acetolactate decarboxylase (ALDC) catalyses a-acetolactate into acetoin (3-hydroxy-2-butanone, AC) and is considered to be the rate-limiting enzyme in the synthesis of 2,3-butanediol. In this work, the enzymatic activity of ALDC from Enterobacter aerogenes ALDC (E.a.-ALDC) was fully characterized with enzyme kinetics, indicating a K-m of 14.83 +/- 0.87 mM and a k(cat) of 0.81 +/- 0.09 s(-1). However, compared with the activities of ALDCs reported from other bacteria, the activity of E.a.-ALDC was determined to present a relatively lower value of 849.08 +/- 35.21 U/mg. The enzyme showed maximum activity at pH 5.5. In addition, the activity of Ea-ALDC was promoted by Mg2+. The crystal structure of E.a.-ALDC firstly solved by X-ray crystallography at resolution of 2.4 angstrom revealed a chelated zinc ion with conserved His199, His201, His212, G1u70 and G1u259. In the active center, the conservative Arg150 was particularly proven to deviate from the zinc ion of the active centre, by adopting a flexible conformational change, resulting in a weak interaction network of the enzyme and the substrate. Further in silico docking of E.a.-ALDC with two enantiomers, (R)-acetolactate and (S)-acetolactate, unaltered the interaction network of E.a.-ALDC from the apo structure, which confirmed the weakened role of Arg150 in the catalytic properties of E.a.-ALDC. Our results reveal a unique structure-function relationship of acetolactate decarboxylase and provide a fundamental basis for the enzymatic synthesis of acetoin.