Role of serine 352 in the allosteric response of Serratia marcescens aspartokinase I-homoserine dehydrogenase I analyzed by using site-directed mutagenesis

Abstract

Aspartokinase I and homoserine dehydrogenase I (AKI-HDI) from Serratia marcescens Sr4l are encoded by the thrA gene as a single polypeptide chain. Previously, a single amino acid substitution of Ser-352 with Phe was shown to produce an AKI-HDI enzyme that is not subject to threonine-mediated feedback inhibition. To determine the role of Ser-352 in the allosteric response, the thrA gene was modified by using site-directed homoserine dehydrogenase II and contains the thrA1SA25 mutation. These strains produce 4 to 24 g of L-threonine per liter in a fermentation medium containing sucrose and urea (10)(11)(12)24). Both AKI and HDI of the strains HNr21 and TLr156 are desensitized for threonine-mediated feedback inhibition (10,24). Strain HNr59 carries threonine-sensitive AKI and threonine-resistant HDI (12).The mutations of these threonine-producing strains have been revealed to result from a single amino acid substitution in the Among these three substitutions, the amino acid replacement of Ser-352 with Phe is the most effective for the desensitization for threonine-mediated feedback inhibition. This paper deals with the changes in the allosteric response of AKI and HDI activities resulting from amino acid substitutions at position 352 in the AKI-HDI protein. MATERUILS AND METHODSBacterial strains and plasmids. The strains and plasmids used are listed in Table 1. E. coli JM109 was used as a host 959 on April 27, 2019 by guest

Characterization of theL-Lysine Biosynthetic Pathway in the Obligate MethylotrophMethylophilus methylotrophus

“…The bacterial strains and plasmids used in this study are listed in Table 1. 20) and 10 ml 37 C or in M9 medium 21) supplemented with 50 mg/l of diaminopimelate (DAP), L-threonine, and L-methionine, depending on the situation. Appropriate antibiotics were added as previously described.…”

“…The mutation in G49 AK was in the region involved in the allosteric regulation of AK. 37) With respect to DDPS, the predicted amino acid sequence of the M. methylotrophus product had identities of 79% with that of M. glycogenes, 38) also an obligate methylotroph, and 55% with that of E. coli (292 aa), and relatively low identities of 45%, 39%, and 30% with those of B. subtilis (290 aa), C. glutamicum (301 aa), and P. aeruginosa (293 aa) respectively. A comparison of the amino acid sequences of the five dapA genes is shown in Fig.…”

“…Strain AS1 was grown for 16 h in SEIIa medium to a density of 10 8 cells/ml, harvested, washed, and resuspended at 10 9 cells/ml in 50 mM potassium phosphate buffer (pH 7.0), containing 100 mg/l N-methyl-N 0 -nitro-N-nitrosoguanidine (MNNG). After being incubated at 37 C for varying durations, cells were harvested and washed, and the cell pellet was resuspended in fresh SEIIa medium and grown overnight. The cells were then spread on SEIIa agar medium containing 7 g/l AEC and 3 g/l L-threonine, and incubated for 3 to 4 d at 37 C.…”

“…After being incubated at 37 C for varying durations, cells were harvested and washed, and the cell pellet was resuspended in fresh SEIIa medium and grown overnight. The cells were then spread on SEIIa agar medium containing 7 g/l AEC and 3 g/l L-threonine, and incubated for 3 to 4 d at 37 C.…”

“…M. methylotrophus was grown at 37 C for 16 h in SEIIa medium until it reached late-log phase. The cells were harvested, washed, and suspended in buffer B (50 mM potassium phosphate, pH 7.5; 30 mM 2-mercaptoethanol; and 10 mM MgSO 4 ).…”