Naoki Eisaki

Effects of bienzyme complex formation of cysteine synthetase from escherichia coli on some properties and kinetics.

Some properties and kinetics of the free and bound serine acetyltransferases (SATs) and O-acetylserine sulfhydrylase-As (OASS-As) from Escherichia coli were investigated. In some cases, SATΔC20, deleting 20 amino acid residues from the C-terminus of the wild-type SAT (Biosci. Biotechnol. Biochem., 63, 168-179 (1999)) was tested for comparison. The optimum pH and stability against some reagents for the free and bound wild-type SATs were similar except for the resistance to cold inactivation. The kinetics for the wild-type SAT and SATΔC20 followed a Ping-Pong Bi Bi mechanism with a mixed-type inhibition by L-cysteine. The kinetics and kinetic constants for the wild-type SAT were not changed by the complex formation with OASS-A. The optimum pH for OASS-A was shifted towards an alkaline pH by the complex formation. Thermal stability and stability against some reagents for the free and bound OASS-As were almost the same. On the other hand, the maximum velocity for OASS-A was lowered and dissociation constants for the substrates and products were increased by forming the complex with the wild-type SAT, although the kinetics for the free and bound enzymes followed the same Ping-Pong Bi Bi mechanism. From comparisons of computed courses of L-cysteine formation from L-serine using SAT (wild-type SAT and SATΔC20) and OASS-A with the experimental results and changes in the stability of the wild-type SAT by the complex formation, we discuss the role and significance of a complex formation for the cysteine synthetase.

Characteristics of serine acetyltransferase from Escherichia coli deleting different lengths of amino acid residues from the C-terminus.

Some properties of serine acetyltransferases (SATs) from Escherichia coli, deleting 10-25 amino acid residues from the C-terminus (SATΔC10-ΔC25) were investigated. The specific activity depended only slightly on the length of the C-terminal region deleted. Although the sensitivity of SATΔC10 to inhibition by L-cysteine was similar to that for the wild-type SAT, it became less with further increases in the length of the amino acid residues deleted. SATΔC10 was inactivated on cooling to 0°C and dissociated into dimers or trimers in the same manner as the wild-type SAT, but Met-256-Ile mutant SAT as well as SATΔC14, SATΔC20, and SATΔC25 were stable. Since SATΔC10, SATΔC14, and SATΔC25 did not form a complex with O-acetylserine sulfhydrylase-A (OASS-A) in a way similar to SATΔC20, it was indicated that 10 amino acid residues or fewer from the C-terminus of the wild-type SAT are responsible for the complex formation with OASS-A. The C-terminal peptide of the 10 amino acid residues interacted competitively with OASS-A with respect to OAS although its affinity was much lower than that for the wild-type SAT.

Increase in the stability of serine acetyltransferase from Escherichia coli against cold inactivation and proteolysis by forming a bienzyme complex.

Cysteine synthetase from Escherichia coli is a bienzyme complex composed of serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase-A (OASS). The effects of the complex formation on the stability of SAT against cold inactivation and proteolysis were investigated. SAT was reversibly inactivated on cooling to 0°C. Ultracentrifugal analysis showed that SAT (a hexamer) was dissociated mostly into two trimers on cooling to 0°C in the absence of OASS, while in the presence of OASS one trimer of the SAT subunits formed a complex with one dimer of OASS subunits. In the presence of OASS, not only the cold inactivation rate was reduced but also the reactivation rate was increased. Furthermore, SAT became stable against proteolytic attack by α-chymotrypsin and V8 protease by forming the complex with OASS. On the other hand, SAT was degraded by trypsin in the same manner both in the presence and in the absence of OASS. The different tendency in the stability against proteolysis with the different proteases was discussed with respect to the substrate specificity of the proteases and amino acid sequence of the C-terminal region of SAT that interacts with OASS.

Pyruvate phosphate dikinase from a thermophilic actinomyces Microbispora rosea subsp. aerata: purification, characterization and molecular cloning of the gene.

Various thermophilic bacteria were analyzed by Southern hybridization analysis using oligonucleotide probes coding for the pyruvate phosphate dikinase (PPDK) gene from Clostridium symbiosum, and positive hybridization signals were observed in the chromosomal DNAs from Microbispora rosea subsp. aerata (IFO 14047). PPDK activity was detected in lactose induced cells and the enzyme was purified to homogeneity. The molecular mass of PPDK was estimated to be 230000 by gel filtration chromatography and 91000 by SDS-PAGE, suggesting that PPDK is a dimeric enzyme. This enzyme was specific for adenine nucleotide and the apparent Km values for AMP, PPi, and phosphoenolpyruvate were 5, 38, and 280 microM, respectively. It was stable in the pH range 6 to 11, and retained 80% activity after 60 min heat treatment at 60 degrees C. We cloned the PPDK gene from M. rosea. It consists of 878 amino acids with a molecular mass of 95514. Sequence comparison indicates around 50% similarity with other PPDKs and it has all the highly conserved regions of the related enzymes. We expressed the PPDK gene in Escherichia coli and obtained enzymatically active protein.

Production of β-(pyrazol-1-yl)-l-alanine from l-serine and pyrazol using recombinant Escherichia coli cells expressing serine acetyltransferase and O-acetylserine sulfhydrylase-A

Abstractβ-(Pyrazol-1-yl)-l-alanine (β-PA) was produced from l-serine and pyrazol using recombinant Escherichia coli cells expressing serine acetyltransferase and O-acetylserine sulfhydrylase-A. The amount of β-PA increased with increasing l-serine concentrations up to 600 mM at 50 mM pyrazol while 100 mM pyrazol gave the highest β-PA production with 50 mM l-serine. Under the optimized conditions, β-PA accumulated in the broth at approximately 140 mM with a conversion of 90% with respect to the added amount of pyrazol.

Development of the Two-Stage Culture System for Production of Useful Compounds Through Simultaneous Gene-Amplification

Abstract We have constructed the recombinant phage harboring the genes that code for enzymes for cysteine biosynthesis with acetylCoA regeneration system by using the Sleeper system that we have developed originally. We designed a two-stage culture system in which the stage between for growth and for enzyme production can be separated through a heat exchanger and the culture conditions for each stage can be controlled independently, combined with the Sleeper system. Fermentation performance of the lysogen that harbors the genes for cysteine biosynthesis in the two-stage culture system was compared with the single-stage culture system. In the two-stage culture system we have designed the serine acetyltransferase(SAT) activity reached to 2.5U/mg-protein while lU/mg-protein in case of the single-stage culture.