The three-dimensional structures of two forms of the D-amino acid aminotransferase (D-aAT) from Bacillus sp. YM-1 have been determined crystallographically: the pyridoxal phosphate (PLP) form and a complex with the reduced analogue of the external aldimine, N-(5'-phosphopyridoxyl)-d-alanine (PPDA). Together with the previously reported pyridoxamine phosphate form of the enzyme [Sugio et al. (1995) Biochemistry 34, 9661], these structures allow us to describe the pathway of the enzymatic reaction in structural terms. A major determinant of the enzyme's stereospecificity for D-amino acids is a group of three residues (Tyr30, Arg98, and His100, with the latter two contributed by the neighboring subunit) forming four hydrogen bonds to the substrate alpha-carboxyl group. The replacement by hydrophobic groups of the homologous residues of the branched chain L-amino acid aminotransferase (which has a similar fold) could explain its opposite stereospecificity. As in L-aspartate aminotransferase (L-AspAT), the cofactor in D-aAT tilts (around its phosphate group and N1 as pivots) away from the catalytic lysine 145 and the protein face in the course of the reaction. Unlike L-AspAT, D-aAT shows no other significant conformational changes during the reaction.

NAD-and glutathione-dependent formaldehyde dehydrogenase (GD-FALDH) ofParacoccus denitrificans has been purified as a tetramer with a relative molecular mass of 150 kDa. The gene encoding GD-FALDH (flhA) has been isolated, sequenced, and mutated by insertion of a kanamycin resistance gene. The mutant strain is not able to grow on methanol, methylamine, or choline, while heterotrophic growth is not influenced by the mutation. This finding indicates that GD-FALDH of P. denitrificans is essential for the oxidation of formaldehyde produced during methylotrophic growth.Paracoccus denitrificans is a gram-negative, aerobic soil bacterium which is able to grow on methanol, methylamine, and choline. The oxidation of methanol and methylamine to formaldehyde is catalyzed by the periplasmically located enzymes methanol dehydrogenase and methylamine dehydrogenase, respectively. During growth on methylamine, formaldehyde is transported to the cytoplasm by a transport mechanism in which a transport protein is involved (16). During the oxidation of choline to glycine, several molecules of formaldehyde are produced. In the presence of formaldehyde and reduced glutathione, the compound S-hydroxymethylglutathione is nonenzymatically formed. NAD-and glutathione-dependent formaldehyde dehydrogenase (GD-FALDH) oxidizes S-hydroxymethylglutathione to S-formylglutathione (14,19,20,24,27), which is oxidized further via formate to carbon dioxide. In this report, we describe the purification of GD-FALDH of P. denitrificans and the isolation and mutagenesis of the gene encoding this enzyme. From growth characteristics of the mutant strain, it can be concluded that GD-FALDH of P. denitrificans is essential for methylotrophic growth.Purification and biochemical analysis of GD-FALDH. GD-FALDH was purified approximately 50-fold from methylamine-grown P. denitrificans cells, as shown in Table 1. By this method, 65 g (wet weight) of cells was harvested and washed with 50 mM Tris hydrochloride (pH 7.5). The cells were disrupted with a French pressure cell, yielding the cell extract. Enzyme activity was measured routinely by determining the rate of NADH formation at 340 nm at room temperature. The assay mixture contained (final concentrations) 0.1 M Na 4 P 2 O 7 -HCl (pH 9.0), 2 mM (reduced) glutathione, and 2.5 mM NAD. After 30 s of incubation with enzyme solution, the reaction was started by adding formaldehyde to a final concentration of 5 mM. Activities were calculated by using a molar absorption coefficient for NADH at 340 nm of 6,220 M Ϫ1 cm Ϫ1 (4).(NH 4 )SO 4 was added to the cell extract. The enzyme precipitated between 25 and 60% saturation. The precipitate was dissolved in 10 mM potassium phosphate buffer (KPB; pH 7.0) and applied to a Phenyl-Sepharose HP column (12.4 by 2.6 cm) equilibrated with 1.5 M (NH 4 )SO 4 in 10 mM KPB (pH 7.0). After washing of the column with the same buffer, elution occurred with a gradient of 1.5 to 0 M (NH 4 )SO 4 in 10 mM KPB in 3 h at a flow rate 3 ml/min. GD-FALDH eluted when the (NH 4 )SO 4 concentration ...

NAD-linked, factor-dependent formaldehyde dehydrogenase (FD-FA1 DH) of the Gram-positive methylotrophic bacterium, Amycolatopsis methanolica, was purified to homogeneity. It is a trimeric enzyme with identical subunits (molecular mass 40 kDa) containing 6 atoms Zn/enzyme molecule. The factor is a heat-stable, low-molecular-mass compound, which showed retention on an Aminex HPX-87H column. Inactivation of the factor occurred during manipulation, but activity could be restored by incubation with dithiothreitol. The identity of the factor is still unknown. It could not be replaced by thiol compounds or cofactors known to be involved in metabolism of C1 compounds. Of the aldehydes tested, only formaldehyde was a substrate. However, the enzyme showed also activity with higher aliphatic alcohols and the presence of the factor was not required for this reaction. Methanol was not a substrate, but high concentrations of it could replace the factor in the conversion of formaldehyde. Presumably, a hemiacetal of formaldehyde is the genuine substrate, which, in the case of methanol, acts as a factor leading to methylformate as the product. This view is supported by the fact that formate could only be detected in the reaction mixture after acidification. Inhibition studies revealed that the enzyme contains a reactive thiol group, being protected by the binding of NAD against attack by heavy-metal ions and aldehydes. Studies on the effect of the order of addition of coenzyme and substrate suggested that optimal catalysis required NAD as the first binding component. Substrate specificity and the induction pattern clearly indicate a role of the enzyme in formaldehyde oxidation. However, since FD-FAlDH was also found in A . methanolica grown on n-butanol, but not on ethanol, it may have a role in the oxidation of higher aliphatic alcohols as well. FD-FA1DH and the factor from A . methanolica are very similar to a combination already described for Rhodococcus erythropolis [Eggeling, L. & Sahm, H. (1985) Eur. J . Biochem. 1-50, 129-1341. NAD-linked, glutathione-dependent formaldehyde dehydrogenase (GD-FA1 DH) resembles FD-FAIDH in many respects. Since glutathione has so far not been detected in Gram-positive bacteria, FD-FA1DH could be the counterpart of this enzyme in Gram-positive bacteria. Alignment of the Nterminal sequence (31 residues) of FD-FAlDH with that of GD-FAlDH from rat liver indeed showed similarity (30% identical positions). However, comparable similarity was found with class I alcohol dehydrogenase from this organism and with cytosolic alcohol dehydrogenase from Succharomyces cerevisiae, isozyme 1. Therefore, it is concluded that the trimeric FD-FA1 DH is related to the dimeric/ tetrameric, zinc-containing, long-chain alcohol dehydrogenases. ~~Corre.pindence to J. A. Duine,

d-amino acid aminotransferase (d-aAT, EC 2.6.1.21) is a pyridoxal-phosphate (PLP) dependent enzyme that specifically transaminates d-amino acids. d-aAT provides one of the routes for the biosynthesis of d-alanine and/or d-glutamate, which are essential constituents of the bacterial cell wall peptidoglycan, thereby making this enzyme a potential antimicrobial target. One agent that inhibits this enzyme is d-cycloserine, believed to react with the cofactor and subsequently form a covalent link to the protein. We have recently reported the high-resolution crystal structure of d-aAT from a thermophilic Bacillus species (Sugio et al. Biochemistry 1995, 34, 9961−9969). We now report the crystal structure (PDB accession code 2DAA) of this enzyme inactivated by d-cycloserine. Contrary to expectations, cycloserine is not covalently attached to the protein but rather forms a stable aromatic species attached to the cofactor and held in place by many noncovalent interactions. The chemical nature of the complex between d-aAT and cycloserine was confirmed by infrared and nuclear magnetic resonance spectroscopy. This observation sheds light not only on the mechanism of inhibition of PLP-dependent aminotransferases by cycloserine in general but also on the nature of substrate recognition by d-aAT.