Nucleotide sequence of the Serratia marcescens threonine operon and analysis of the threonine operon mutations which alter feedback inhibition of both aspartokinase I and homoserine dehydrogenase I

Abstract

The nucleotide sequence of the Serratia narcescens threonine operon (thrA$,2BC) was determined. Three long open reading frames were identified; these open reading frames code for aspartokinase I (AKI)-homoserine dehydrogenase I (HDI), homoserine kinase, and threonine synthase, in that order. The predicted amino acid sequences of these enzymes were similar to the amino acid sequences of the corresponding enzymes in Escherichia coli. The AKI-HDI protein is apparently a tetramer composed of monomer polypeptides that are 819 amino acids long. A deletion analysis revealed that the central and C-terminal region was responsible for threonine-resistant HDI activity, a monomeric fragment extending from the N terminus to residue 306 was responsible for threonine-resistant AKI activity, and an N-terminal portion containing 468 residues was responsible for threonine-sensitive AKI activity. The thrAlIA21 and thrA15A25 mutations of threonineexcreting strains HNr21 and TLr156, which result in the loss of threonine-mediated feedback inhibition of both AKI activity and HDI activity, cause single amino acid substitutions (Gly to Asp at position 330 and Ser to Phe at position 352, respectively) in the central region of the AKI-HDI protein. The thrA1+A22 mutation of strain HNr59, which results in a threonine-sensitive AKI and a threonine-resistant HDI, also causes a single amino acid substitution (Ala to Thr at position 479).Threonine is synthesized from L-aspartate via five enzymatic reactions. In Escherichia coli, four of the five enzyme activities of this pathway are encoded by the thrAlA2BC operon (39,45). The first and third steps of this pathway are catalyzed by aspartokinase I (AKI) (EC 2.7.2.4) and homoserine dehydrogenase I (HDI) (EC 1.1.1.3); these two enzymes are encoded by the thrA A2 genes in a single polypeptide chain, the AKI-HDI protein. Homoserine kinase (EC 2.7.1.39), the thrB gene product, catalyzes the fourth step. The thrC gene encodes threonine synthase (EC 4.2.99.2), which catalyzes the last step of the pathway. The nucleotide sequences of these thr genes have been reported previously (6,19,35). The E. coli AKI-HDI enzyme is a large tetrameric protein (10,19). Proteolytic digestion has shown that the AKI and HDI activities are carried by the N-terminal and C-terminal moieties, respectively, and that multimer formation depends on the central region (11,12).Threonine synthesis in Serratia marcescens has been studied extensively (22,23). Both AKI and HDI activities are subject to feedback inhibition by threonine, and expression of the thr operon is multivalently repressed by threonine and isoleucine. Therefore, the AKI-HDI protein is a key enzyme of this pathway in S. marcescens.Threonine-producing isolates of S. marcescens Sr4l were obtained previously, and these organisms were found to be insensitive to feedback inhibition for HDI or for both AKI and HDI. Strains HNr21 and HNr59 are resistant to a threonine analog, 0-hydroxynorvaline (23). Both AKI activity and HDI activity in strain HNr21 are resistant to thr...

Characterization of mutations induced by N-methyl-N′-nitro-N-nitrosoguanidine in an industrial Corynebacterium glutamicum strain

“…Some of these mutants have been shown to be genetically deregulated with respect to relevant 2 biosynthetic pathways. However, recent more detailed analysis has revealed that mutations responsible for deregulation, such as the mutations in thrA [4], dapA [5], lysC [6], and gnd [7], resulted in only partial desensitization of the enzymes, despite continual efforts of strain improvement. This made us realize again that NTG mutagenesis is not necessarily the best to achieve high desensitization of regulatory enzymes.…”

Characterization of an Arabidopsis thaliana cDNA encoding an S‐adenosylmethionine‐sensitive threonine synthase Threonine synthase from higher plants

“…The corresponding gene has been isolated from a number of bacteria [6][7][8][9][10][11] and from Saccharomyces cerevisiae [12]. In plants, only partial purification of threonine synthase has been achieved [13 15].…”

A new mutation in the yeast aspartate kinase induces threonine accumulation in a temperature‐regulated way

“…The ACT domain has been found in enzymes that are regulated by amino acids or purines and is involved in regulation by these effectors (Chipman and Shaanan, 2001). In Figure 5, only aspartate kinases for which single mutations affecting enzymatic activity or regulation have been described (Arévalo-Rodríguez et al, 1999;Bareich and Wright, 2003;Farfán, 1997;Follettie et al, 1993;Hernando-Rico et al, 2001;Kalinowski et al, 1991;Kikuchi et al, 1999;Marco-Marin et al, 2003;Martín-Rendón et al, 1993;Ogawa-Miyata et al, 2001;Ohnishi et al, 2002;Omori et al, 1993;Paris et al, 2003) are shown. Most but not all of those mutations affecting regulation are located in the ACT domains.…”

“…A few of them are in the amino acid kinase domain, viz. E282D and E279A in S. cerevisiae (this work; Bareich and Wright, 2003) (12) I522A (12) Q524 (12) AtAK-HD S301Y (8) G345D (8) AlAK E164K (6) K8R (5) E119D (5) R198K (5) D202E (5) E250K (7) G323D (7,6) M318I (6) L325F (6) T352I (7,6) V347M (6) T344M (7) S345L (7,6) EcAKIII S301Y (9) T311Y ( Bareich and Wright, 2003;(2) Arévalo-Rodríguez et al, 1999;(3) Martín-Rendón et al, 1993;(4) Farfán, 1997;(5) Marco-Marin et al, 2003;(6) Ogawa-Miyata et al, 2001;(7) Kikuchi et al, 1999;(8) Hernando-Rico et al, 2001;(9) Kalinowski et al, 1991;(10) Ohnishi et al, 2002;(11) Follettie et al, 1993;(12) Paris et al, 2003;(13) Omori et al, 1993Omori et al, et al, 1999Marco-Marin et al, 2003;O...…”