Histidine carboxylase of Leuconostoc nos 9204: purification, kinetic properties, cloning and nucleotide sequence of the hdc gene

摘要

Histidine decarboxylase (HDC) was purified to homogeneity form Leuconostoc nos 9204, a wine lactic acid bacterium. Histidine decarboxylase comprised two subunits, respectively α and β. The hdc gene was cloned and sequenced. The gene encodes a single polypeptide of 315 amino acids, demonstrating that Leuc. nos 9204 HDC was synthesized as a precursor proHDC π_6 (Mr 205 000). A cleavage between Ser-81 and Ser-82 generated the α(Mr 25 380) and β(Mr 8840) chains, which suggested that the holoenzyme exists as a hexameric structure (αβ)_6. At the optimal pH of 4.8, the HDC activity exhibited a simple Michaelis-Menten kinetic (K_m = 0.33 mmol 1~(-1), V_(max) = 17.8 μmol CO_2 min~(-1) mg~(-1)), while at pH 7.6 it was sigmoidal (cooperativity index of 2). Histamine acted as a competitive inhibitor (K_i = 32 mmol 1~(-1)). The similarities of these results with those described for other bacterial HDC support the assumption that the pyruvoyl enzymes evolved from a common ancestral protein and have similar catalytic mechanisms. These results also confirmed that the main lactic acid bacterial species responsible for malolactic fermentation in red wine is able to produce histamine. Bacteria carrying the HDC activity must be avoided during selection f strains for the production of malolactic starters.