Isolation and characterization of a benzoylformate decarboxylase and a NAD+/NADP+-dependent benzaldehyde dehydrogenase involved in D-phenylglycine metabolism in Pseudomonas stutzeri ST-201.
ABSTRACT Following induction with D-phenylglycine both d-phenylglycine aminotransferase activity and benzoylformate decarboxylase activity were observed in cultures of Pseudomonas stutzeri ST-201. Induction with benzoylformate, on the other hand, induced only benzoylformate decarboxylase activity. Purification of the benzoylformate decarboxylase, followed by N-terminal sequencing, enabled the design of probes for hybridization with P. stutzeri ST-201 genomic DNA libraries. Sequencing of two overlapping genomic DNA restriction fragments revealed two open reading frames which were denoted dpgB and dpgC. Sequence alignments suggested that the genes encoded a thiamin-diphosphate-dependent decarboxylase and an aldehyde dehydrogenase, respectively. Both genes were isolated and expressed in Escherichia coli. The dpgB gene product was confirmed as a benzoylformate decarboxylase while the dpgC gene product was characterized as a NAD+/NADP+-dependent benzaldehyde dehydrogenase. In keeping with their high sequence identities (both greater than 85%) the kinetic properties of the two enzymes were similar to those of the homologous enzymes in the mandelate pathway of Pseudomonas putida ATCC 12633. However, Pseudomonas stutzeri ST-201 was unable to grow on either isomer of mandelate, and sequencing indicated that the dpgB gene did not form part of an operon. Thus it appears that the two enzymes form part of a d-phenylglycine, rather than mandelate, degrading pathway.
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ABSTRACT: A new large-scale purification method for benzoylformate decarboxylase from Pseudomonas putida has allowed us to undertake an X-ray crystallographic study of the enzyme. The previously observed instability of the enzyme was overcome by addition of 100 μM thiamine pyrophosphate to buffers used in the purification. The final enzyme preparation was more than 97% pure, as determined by denaturing gel electrophoresis and densitometry. The mobility of the enzyme on a gel filtration column indicates that it is a tetramer of 57-kDa subunits. Large, single crystals of benzoylformate decarboxylase were grown from solutions of buffered polyethylene glycol 400, pH 8.5. The crystals diffract to beyond 1.6 Å resolution and are stable for days to X-ray radiation. Analysis of X-ray data from the crystals, along with the newly determined quaternary structure, identifies the space group as 1222. The unit cell dimensions are a = 82 Å, b = 97 Å, c = 138 Å. An average Vm value for the crystals is consistent with one subunit per asymmetric unit. The subunits of the tetramer must be arranged with tetrahedral 222 symmetry.Protein Science 04/1995; 4(5):955 - 959. · 2.74 Impact Factor
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ABSTRACT: D-phenylglycine aminotransferase (D-PhgAT) from a newly isolated soil bacterium, Pseudomonas stutzeri ST-201, was purified to electrophoretic homogeneity and characterized. The molecular weight (M(r)) of the native enzyme was estimated to be 92,000. It is composed of two subunits identical in molecular weight (M(r)) = 47,500). The isoelectric point (pI) of the native enzyme was 5.0. The enzyme catalyzed reversible transamination specific for D-phenylglycine or D-4-hydroxyphenylglycine in which 2-oxoglutarate was an exclusive amino group acceptor and was converted into L-glutamic acid. Neither the D- nor L-isomer of phenylalanine, tyrosine, alanine, valine, leucine, isoleucine or serine could serve as a substrate. The enzyme was most active at alkaline pH with maximum activity at pH 9-10. The temperature for maximum activity was 35-45 degrees C. The apparent K(m) values for D-phenylglycine and for 2-oxoglutarate at 35 degrees C, pH 9.5 were 1.1 and 2.4 mM, respectively. The enzyme activity was strongly inhibited by typical inhibitors of pyridoxal phosphate-dependent enzymes. Possible application of this enzyme for synthesis of enantiomerically pure D-phenylglycine was demonstrated.Journal of Biotechnology 08/1997; 55(3):193-203. · 3.18 Impact Factor
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ABSTRACT: Thiamin diphosphate (ThDP)-dependent enzymes catalyze a range of transformations, such as decarboxylation and ligation. We report a novel spectroscopic assay for detection of some of the ThDP-bound intermediates produced on benzoylformate decarboxylase. Benzoylformate decarboxylase was mixed with its alternate substrate p-nitrobenzoylformic acid on a rapid-scan stopped-flow instrument, resulting in formation of three absorbing species (lambda(max) in parentheses): I(1) (a transient at 620 nm), I(2) (a transient at 400 nm), and I(3) (a stable absorbance with lambda(max) > 730 nm). Analysis of the kinetics of the two transient species supports a model in which a noncovalent complex of the substrate and the enzyme is converted to the first covalent intermediate I(1); the absorbance corresponding to I(1) is probably a charge-transfer band arising from the interaction of the thiamin diphosphate-p-nitrobenzoylformic acid covalent adduct (2-p-nitromandelylThDP) and the enzyme. The rate of disappearance of I(1) parallels the rate of formation of I(2). Chemical models suggest the lambda(max) of I(2) (near 400 nm) to be appropriate to the enamine, a key intermediate in ThDP-dependent reactions resulting from the decarboxylation of the thiamin diphosphate-p-nitrobenzoylformic acid covalent adduct. Therefore, the rate of disappearance of I(1) and/or the appearance of I(2) directly measure the rate of decarboxylation. A relaxation kinetic treatment of the pre-steady-state kinetic data also revealed a hitherto unreported facet of the mechanism, alternating active-sites reactivity. Parallel studies of the His70Ala BFD active-site variant indicate that it cannot form the complex reported by the charge-transfer band (I(1)) at the level of the wild-type protein.Biochemistry 11/2000; 39(45):13862-9. · 3.38 Impact Factor