Studies on function and regulation of amino acid biosynthetic enzymes
Ayako Yoshida (Biotechnology Research Center, The University of Tokyo)
uayoshi@mail.ecc.u-tokyo.ac.jp
In this study, we focused on the biosynthetic pathways of amino acids and revealed themechanisms of enzyme regulation and the novel biosynthetic machinery by using X-ray crystallography
and biochemical analyses.
Aspartate kinase (AK) is an enzyme catalyzing the first step of lysine biosynthetic pathway
and is regulated by the end-product in a feedback manner. AK from Corynebacterium glutamicum(CgAK), which has been used in the industrial production of lysine, is inhibited in the presence of bothlysine and threonine. Since this regulation of CgAK is a disadvantage for the over-production of lysine,
the mutant strain insensitive to the feedback inhibition has been developed and used. However, themechanism of feedback inhibition had not been elucidated for a long time. Then, we determined thecrystal structures of CgAK in an inhibitory form with inhibitors, and an active form with threonine, andalso a structure of the mutant resistant to the feedback inhibition. The structural comparison clarified
that the binding of inhibitors stabilizes the “closed” inhibitory form and that inhibitor-binding could notstabilize the “closed” conformation in a feedback-resistant mutant. The information of the regulatory
mechanism of CgAK obtained in this study will contribute to more efficient production of lysine.
In a thermophilic bacterium, Thermus thermophilus uses a unique pathway for lysine
biosynthesis, which uses an amino-group carrier protein named LysW. The amino group of αaminoadipate (AAA), an intermediate in this pathway, is protected by the formation of an isopeptide
bond with C-terminal glutamate residue in LysW. We determined the crystal structures of LysW
conjugated with AAA, and LysZ, which is an amino acid kinase, in complex with LysW. As a result,negatively charged LysW interacted with the positively charged area of LysZ, showing that LysW
functions as a protecting-group of the amino-group of AAA and also as a carrier protein, which iselectrostatically recruited to each enzyme, enabling the efficient conversion of AAA to lysine. We also
found that not only lysine but also ornithine could be produced by using LysW system in Thermococcus
kodakarensis where a single set of bifunctional biosynthetic enzymes are involved. This result provided
the experimental evidence for the evolutionary hypothesis, patchwork hypothesis, in metabolic
pathways, suggesting that a pathway composed of multifunctional enzymes was used for the productionof multiple compounds in an ancestral organism with a limited size of genome.
Recently, we are interested in the protein lysine acetylation on the metabolic enzyme, which
is one of the posttranslational modifications of protein known as a new regulatory mechanism ofmetabolism. We conducted the proteomic analysis for T. thermophilus and determined the 208 acetylated
proteins. Among the acetylated proteins identified, we found that 2-isopropylmalate synthase (IPMS)
involved in a leucine biosynthesis is inhibited by the acetylation on a specific lysine residue. Our
observation that the deacetylation of IPMS restored the activity suggested that IPMS is regulated byboth the feedback inhibition by leucine and the protein acetylation. From this study, we provide a novel
regulatory mechanism by the protein acetylation in amino acid biosynthesis.