Toyokazu Yoshida

Crystal structure of a NAD-dependent D-glycerate dehydrogenase at 2.4 Å resolution

D-Glycerate dehydrogenase (GDH) catalyzes the NADH-linked reduction of hydroxypyruvate to D-glycerate. GDH is a member of a family of NAD-dependent dehydrogenases that is characterized by a specificity for the D-isomer of the hydroxyacid substrate. The crystal structure of the apoenzyme form of GDH from Hyphomicrobium methylovorum has been determined by the method of isomorphous replacement and refined at 2.4 A resolution using a restrained least-squares method. The crystallographic R-factor is 19.4% for all 24,553 measured reflections between 10.0 and 2.4 A resolution. The GDH molecule is a symmetrical dimer composed of subunits of molecular mass 38,000, and shares significant structural homology with another NAD-dependent enzyme, formate dehydrogenase. The GDH subunit consists of two structurally similar domains that are approximately related to each other by 2-fold symmetry. The domains are separated by a deep cleft that forms the putative NAD and substrate binding sites. One of the domains has been identified as the NAD-binding domain based on its close structural similarity to the NAD-binding domains of other NAD-dependent dehydrogenases. The topology of the second domain is different from that found in the various catalytic domains of other dehydrogenases. A model of a ternary complex of GDH has been built in which putative catalytic residues are identified based on sequence homology between the D-isomer specific dehydrogenases. A structural comparison between GDH and L-lactate dehydrogenase indicates a convergence of active site residues and geometries for these two enzymes. The reactions catalyzed are chemically equivalent but of opposing stereospecificity. A hypothesis is presented to explain how the two enzymes may exploit the same coenzyme stereochemistry and a similar spatial arrangement of catalytic residues to carry out reactions that proceed to opposite enantiomers.

L-serine production by a methylotroph and its related enzymes.

The production process of l-serine from methanol and glycine has been developed using a methylotroph with the serine pathway. Consecutive reactions of two enzymes, methanol dehydrogenase (MDH) and serine hydroxymethyltransferase (SHMT) are involved in the production. We screened a high producer, Hyphomicrobium methylovorum, which is an obligate methylotroph. With resting cells of the bacterium, 24 mg/ml of l-serine was produced from 100 mg/ml of glycine and 48 mg/ml of methanol in 3 days under optimal conditions. Next, a glycine-resistant mutant GM2 showed improved serine production (32–34 mg/ml). The mutant GM2 was found to have elevated activities of MDH and SHMT. Since there has so far been little report on the systematic characterization of enzymes of the serine pathway in methylotrophs, not only the above two enzymes but also the other three enzymes in H. methylovorum were purified and characterized: MDH, SHMT and hydroxypyruvate reductase (HPR) were crystallized; serine-glyoxylate aminotransferase (SGAT) and glycerate kinase (GK) were purified to homogeneity. As a result, all these enzymes were found to be stable against preservation and to exist abundantly in the bacterium. The gene of SHMT was cloned and its deduced amino acid sequence had homology to those of Escherichia coli (55%) and rabbit liver (44%), whereas the enzyme of the bacterium was immunochemically distinguishable from those of microorganisms other than Hyphomicrobium strains and mammalian livers.

Isolation of a Thiamine-binding Protein from Rice Germ and Distribution of Similar Proteins

A thiamine-binding protein was purified from rice germ (Oryza sativa L.) by extraction, salting-out with ammonium sulfate, and column chromatography. From the results of molecular mass, Kd and Bmax values for thiamine-binding, binding specificity for thiamine phosphates and analog, the protein was suggested to be identical to the thiamine-binding protein in rice bran. The thiamine-binding protein w as more efficiently purified from rice germ than from rice bran. The protein was rich in glutamic acid (and/or glutamine) and glycine. The protein did not show immunological similarity to thiamine-binding proteins in buckwheat and sesame seeds. However proteins similar to the thiamine-binding protein from rice germ existed in gramineous seeds. They were suggested to have thiamine-binding activity and to be of the same molecular mass as the thiamine-binding protein.

Accumulation of thiamine and thiamine-binding protein during development of rice seed.

Summary Thiamine and thiamine-binding activity correlatively increased rice seed from 10 to 30 days after flowering of the plant. Since the thiamine-binding protein in rice seed is known to specifically bind to the nonesterified form of thiamine, the protein appeared to be important in the storage of this type of thiamine provided for germination.

l-Serine production using a resting cell system of Hyphomicrobium strains

Abstract Twenty-six strains of methylotrophic hyphomicrobia were examined for their ability to produce l -serine from methanol and glycine in a resting cell reaction. l -Serine productivity of over 5 mg/ml was observed in 7 strains, and Hyphomicrobium sp. NCIB10099 was found to exhibit the highest productivity. Under optimized conditions using this bacterium, 45 mg/ml l -serine was produced from 100 mg/ml glycine and 88 mg/ml methanol in 3 d. The high l -serine degrading activity of the bacterium was entirely suppressed by adding an appropriate amount of CdCl 2 ( ca. 1 mM), resulting in an enhanced conversion ratio of glycine to l -serine ( ca. 100% molar conversion).

Crystallization and preliminary diffraction studies of hydroxypyruvate reductase (D-glycerate dehydrogenase) from Hyphomicrobium methylovorum

Two crystal forms of hydroxypyruvate reductase (D-glycerate dehydrogenase) from the methylotrophic bacterium Hyphomicrobium methylovorum have been grown from ammonium sulphate solutions. One crystal form is triclinic, with unit cell parameters a = 60.4 A, b = 60.5 A, c = 66.3 A, alpha = 102.3 degrees, beta = 113.7 degrees and gamma = 102.7 degrees, suggesting that a dimer (monomer M(r) 38,000) occupies the unit cell. This crystal form diffracts to beyond 2.4 A resolution and is suitable for crystallographic structure analysis.

l-Serine synthesis using the resting Hyphomicrobium sp. NCIB10099 cells under suppressive conditions for l-serine-degrading activity

Abstract l -Serine-degrading activity could be suppressed by controlling the methanol concentration in l -serine synthesis using resting Hyphomicrobium sp. NCIB10099 cells. Fifty-three mg/ml of l -serine were produced from 100 mg/ml of glycine and 104 mg/ml of methanol. It is believed that l -serine-degrading activity contributes to the reverse serine hydroxymethyltransferase reaction.

Structure and function of the serine pathway enzymes in Hyphomicrobium

A certain group of methylotrophic bacteria possess the serine pathway for the assimilation of C1 compounds, and this pathway operates to synthesize 2- phosphoglycerate as a precursor of cell constituents, comprising the following two part. In the first part, 2-phosphoglycerate is synthesized from glyoxylate and formaldehyde, and malyl-CoA is also formed by subsequent enzyme reactions. In the second part of the pathway, malyl-CoA is cleaved to glyoxylate and acetyl-CoA, and another molecule of glyoxylate is generated from acetyl-CoA. Two variants of the serine pathway have been reported (C. Anthony, 1982), in which the route for the generation of glyoxylate from acetyl-CoA is varied. In the icl +-serine pathway, isocitrate lyase is involved in the route of the oxidation of acetyl-CoA to glyoxylate. Another variant, icl --serine pathway, is known in methylotrophic bacteria such as Methylobacterium and Hyphomicrobium strains (C. Anthony, 1982), which have no isocitrate lyase activities, and the route for the generation of glyoxylate has not been elucidated.