Yasuhiro Mihara

X-ray structures of a novel acid phosphatase from Escherichia blattae and its complex with the transition-state analog molybdate.

The structure of Escherichia blattae non‐specific acid phosphatase (EB‐NSAP) has been determined at 1.9 Å resolution with a bound sulfate marking the phosphate‐binding site. The enzyme is a 150 kDa homohexamer. EB‐NSAP shares a conserved sequence motif not only with several lipid phosphatases and the mammalian glucose‐6‐phosphatases, but also with the vanadium‐containing chloroperoxidase (CPO) of Curvularia inaequalis. Comparison of the crystal structures of EB‐NSAP and CPO reveals striking similarity in the active site structures. In addition, the topology of the EB‐NSAP core shows considerable similarity to the fold of the active site containing part of the monomeric 67 kDa CPO, despite the lack of further sequence identity. These two enzymes are apparently related by divergent evolution. We have also determined the crystal structure of EB‐NSAP complexed with the transition‐state analog molybdate. Structural comparison of the native enzyme and the enzyme–molybdate complex reveals that the side‐chain of His150, a putative catalytic residue, moves toward the molybdate so that it forms a hydrogen bond with the metal oxyanion when the molybdenum forms a covalent bond with NE2 of His189.

Phosphorylation of Nucleosides by the Mutated Acid Phosphatase from Morganella morganii

ABSTRACT A novel nucleoside phosphorylation process using the food additive pyrophosphate as the phosphate source was investigated. TheMorganella morganii gene encoding a selective nucleoside pyrophosphate phosphotransferase was cloned. It was identical to theM. morganii PhoC acid phosphatase gene. Sequential in vitro random mutagenesis was performed on the gene by error-prone PCR to construct a mutant library. The mutant library was introduced intoEscherichia coli, and the transformants were screened for the production of 5′-IMP. One mutated acid phosphatase with an increased phosphotransferase reaction yield was obtained. With E. coli overproducing the mutated acid phosphatase, 101 g of 5′-IMP per liter (192 mM) was synthesized from inosine in an 88% molar yield. This improvement was achieved with two mutations, Gly to Asp at position 92 and Ile to Thr at position 171. A decreasedKm value for inosine was responsible for the increased productivity.

Novel Enzymatic Method for the Production of Xylitol from D-Arabitol by Gluconobacter oxydans

Microorganisms capable of producing xylitol from D-arabitol were screened for. Of the 420 strains tested, three bacteria, belonging to the genera Acetobacter and Gluconobacter, produced xylitol from D-arabitol when intact cells were used as the enzyme source. Among them, Gluconobacter oxydans ATCC 621 produced 29.2 g/l xylitol from 52.4 g/l D-arabitol after incubation for 27 h. The production of xylitol was increased by the addition of 5% (v/v) ethanol and 5 g/l D-glucose to the reaction mixture. Under these conditions, 51.4 g/l xylitol was obtained from 52.4 g/l D-arabitol, a yield of 98%, after incubation for 27 h. This conversion consisted of two successive reactions, conversion of D-arabitol to D-xylulose by a membrane-bound D-arabitol dehydrogenase, and conversion of D-xylulose to xylitol by a soluble NAD-dependent xylitol dehydrogenase. Use of disruptants of the membrane-bound alcohol dehydrogenase genes suggested that NADH was generated via NAD-dependent soluble alcohol dehydrogenase.

Optimum culture conditions for the production of cobalt-containing nitrile hydratase by Rhodococcus rhodochrous J1

SummaryWe sought the optimum conditions for production of nitrile hydratase by Rhodococcus rhodochrous J1. The addiiion of both cobalt ions and an aliphatic nitrile or amide as an inducer was indispensable for the appearance of nitrile hydratase activity in R. rhodochrous J1 cells. Crotonamide was an efficient inducer and, moreover, urea was found to be the most powerful inducer for the production of nitrile hydratase. When R. rhodochrous J1 was cultivated under optimal conditions, the enzyme activity in the culture broth and the specific activity was approximately 32,000 and 512 times higher than the initially obtained levels, respectively. The nitrile hydratase formed corresponded to more than 45% of the total soluble protein in urea-induced cells, as judged by quantitative evaluation of the gel track.

A new enzymatic method of selective phosphorylation of nucleosides

Abstract The phosphorylation of inosine in the 5′-position to produce inosine-5′-monophosphate (5′-IMP) was studied in a number of microorganisms from our culture collection using pyrophosphate (PPi) as the phosphate source. Although many of the microorganisms screened were able to phosphorylate inosine, phosphotransferase activity specific to the 5′-position was found to be distributed among the bacteria belonging to the family Enterobacteriacea. Morganella morganii NCIMB10466 was selected for further study of 5′-IMP production. When M. morganii intact cells were taken approximately 0.2 mg/ml wet weight, 6.02 mg/ml (11.4 mM) of 5′-IMP were synthesized from 10 mg/ml (37.3 mM) of inosine and 250 mg/ml (560.0 mM) of tetrasodium pyrophosphate decahydrate in 9 h.

Improving the Pyrophosphate-inosine Phosphotransferase Activity of Escherichia blattae Acid Phosphatase by Sequential Site-directed Mutagenesis

Escherichia blattae acid phosphatase/phosphotransferase (EB-AP/PTase) exhibits C-5′-position selective pyrophosphate-nucleoside phosphotransferase activity in addition to its intrinsic phosphatase. Improvement of its phosphotransferase activity was investigated by sequential site-directed mutagenesis. By comparing the primary structures of higher 5′-inosinic acid (5′-IMP) productivity and lower 5′-IMP productivity acid phosphatase/phosphotransferase, candidate residues of substitution were selected. Then a total of 11 amino acid substitutions were made with sequential substitutions. As the number of substituted amino acid residues increased, the 5′-IMP productivity of the mutant enzyme increased, and the activity of the 11 mutant phosphotransferases of EB-AP/PTase reached the same level as that of Morganella morganii AP/PTase. This result shows that Leu63, Ala65, Glu66, Asn69, Ser71, Asp116, Thr135, and Glu136, whose relevance was not directly established by structural analysis alone, also plays an important role in the phosphotransferase activity of EB-AP/PTase.

Enzymatic Production of L -Alanyl- L -glutamine by Recombinant E. coli Expressing α-Amino Acid Ester Acyltransferase from Sphingobacterium siyangensis

An enzymatic production method for synthesizing L-alanyl-L-glutamine (Ala-Gln) from L-alanine methyl ester hydrochloride (AlaOMe) and L-glutamine (Gln) was developed in this study. The cultivation conditions for an Escherichia coli strain overexpressing α-amino acid ester acyltransferase from Sphingobacterium siyangensis AJ 2458 (SAET) and reaction conditions for Ala-Gln production were optimized. A high cell density culture broth prepared by fed-batch cultivation showed 440 units/mL of Ala-Gln-producing activity. In addition, an Ala-Gln-producing reaction using intact E. coli cells overexpressing SAET under optimum conditions was conducted. A total Ala-Gln yield of 69.7 g/L was produced in 40 min. The molar yield was 67% against both AlaOMe and Gln.