Crystallization and preliminary X-ray crystallographic analysis of biodegradative threonine deaminase (TdcB) from Salmonella typhimurium

Abstract

Biodegradative threonine deaminase (TdcB) catalyzes the deamination of L-threonine to alpha-ketobutyrate, the first reaction in the anaerobic breakdown of L-threonine to propionate. Unlike the biosynthetic threonine deaminase, TdcB is insensitive to L-isoleucine and is activated by AMP. Here, the cloning of TdcB (molecular weight 36 kDa) from Salmonella typhimurium with an N-terminal hexahistidine affinity tag and its overexpression in Escherichia coli is reported. TdcB was purified to homogeneity using Ni-NTA affinity column chromatography and crystallized using the hanging-drop vapour-diffusion technique in three different crystal forms. Crystal forms I (unit-cell parameters a = 46.32, b = 55.30, c = 67.24 A, alpha = 103.09, beta = 94.70, gamma = 112.94 degrees) and II (a = 56.68, b = 76.83, c = 78.50 A, alpha = 66.12, beta = 89.16, gamma = 77.08 degrees) belong to space group P1 and contain two and four molecules of TdcB, respectively, in the asymmetric unit. Poorly diffracting form III crystals were obtained in space group C2 and based on the unit-cell volume are most likely to contain one molecule per asymmetric unit. Two complete data sets of resolutions 2.2 A (crystal form I) and 1.7 A (crystal form II) were collected at 100 K using an in-house X-ray source.

Figure 1

Metabolic pathway illustrating the anaerobic breakdown of l-threonine to propionate, in which the first reaction is catalyzed by biodegradative threonine deaminase (TdcB).

Figure 2

SDS–PAGE analysis of TdcB during purification. Proteins were analysed on 12% SDS–PAGE and stained with Coomassie blue. Lane 1, crude cell lysates after 0.3 mM IPTG induction; lane 2, clear supernatant; lane 3, purified TdcB after Ni–NTA affinity column chromatography; lane M, molecular-weight markers (kDa).

Figure 3

Crystals of TdcB obtained in three different crystal forms in the presence of crystallization conditions containing (a) 2-propanol (crystal form I), (b) t-butanol (crystal form II) and (c) 1,6-hexanediol (crystal form III).