Structure-Based Mechanism for Oxidative Decarboxylation Reactions Mediated by Amino Acids and Heme Propionates in Coproheme Decarboxylase (HemQ).

Coproheme decarboxylase catalyzes two sequential oxidative decarboxylations with H2O2 as the oxidant, coproheme III as substrate and cofactor, and heme b as the product. Each reaction breaks a C-C bond and results in net loss of hydride, via steps that are not clear. Solution and solid-state structural characterization of the protein in complex with a substrate analog revealed a highly unconventional H2O2-activating distal environment with the reactive propionic acids (2 and 4) on the opposite side of the porphyrin plane. This suggested that, in contrast to direct C-H bond cleavage catalyzed by a high-valent iron intermediate, the coproheme oxidations must occur through mediating amino acid residues. A tyrosine that hydrogen bonds to propionate 2 in a position analogous to the substrate in ascorbate peroxidase is essential for both decarboxylations, while a lysine that salt bridges to propionate 4 is required solely for the second. A mechanism is proposed in which propionate 2 relays an oxidizing equivalent from a coproheme compound I intermediate to the reactive deprotonated tyrosine, forming Tyr•. This residue then abstracts a net hydrogen atom (H•) from propionate 2, followed by migration of the unpaired propionyl electron to the coproheme iron to yield the ferric harderoheme and CO2 products. A similar pathway is proposed for decarboxylation of propionate 4, but with a lysine residue as an essential proton shuttle. The proposed reaction suggests an extended relay of heme-mediated e-/H+ transfers and a novel route for the conversion of carboxylic acids to alkenes.

Biochemical and genetic characterization of the Enterococcus faecalis oxaloacetate decarboxylase complex.

Enterococcus faecalis encodes a biotin-dependent oxaloacetate decarboxylase (OAD), which is constituted by four subunits: E. faecalis carboxyltransferase subunit OadA (termed Ef-A), membrane pump Ef-B, biotin acceptor protein Ef-D, and the novel subunit Ef-H. Our results show that in E. faecalis, subunits Ef-A, Ef-D, and Ef-H form a cytoplasmic soluble complex (termed Ef-AHD) which is also associated with the membrane. In order to characterize the role of the novel Ef-H subunit, coexpression of oad genes was performed in Escherichia coli, showing that this subunit is vital for Ef-A and Ef-D interaction. Diminished growth of the oadA and oadD single deletion mutants in citrate-supplemented medium indicated that the activity of the complex is essential for citrate utilization. Remarkably, the oadB-deficient strain was still capable of growing to wild-type levels but with a delay during the citrate-consuming phase, suggesting that the soluble Ef-AHD complex is functional in E. faecalis. These results suggest that the Ef-AHD complex is active in its soluble form, and that it is capable of interacting in a dynamic way with the membrane-bound Ef-B subunit to achieve its maximal alkalinization capacity during citrate fermentation.

Optimization of a non-radioactive high-throughput assay for decarboxylase enzymes.

Herein, we describe the optimization of a linked enzyme assay suitable for high-throughput screening of decarboxylases, a target family whose activity has historically been difficult to quantify. Our approach uses a commercially available bicarbonate detection reagent to measure decarboxylase activity. The assay is performed in a fully enclosed automated screening system under inert nitrogen atmosphere to minimize perturbation by exogenous CO2. Receiver operating characteristic (ROC) analysis following a pilot screen of a small library of approximately 3,600 unique molecules for inhibitors of Trypanosoma brucei ornithine decarboxylase quantitatively demonstrates that the assay has excellent discriminatory power (area under the curve = 0.90 with 95% confidence interval between 0.82 and 0.97).

Purification and molecular cloning of rat 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase.

2-Amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD; EC 4.1.1.45) is one of the important enzymes regulating tryptophan-niacin metabolism. In the present study, we purified the enzyme from rat liver and kidney, and cloned the cDNA encoding rat ACMSD. The molecular masses of rat ACMSDs purified from the liver and kidney were both estimated to be 39 kDa by SDS/PAGE. Analysis of N-terminal amino acid sequences showed that these two ACMSDs share the same sequence. An expressed sequence tag (EST) of the mouse cited from the DNA database was found to be identical with this N-terminal sequence. Reverse transcription-PCR (RT-PCR) was performed using synthetic oligonucleotide primers having the partial sequences of the EST, and then cDNAs encoding rat ACMSDs were isolated by using subsequent 3'-rapid amplification of cDNA ends and RT-PCR methods. ACMSD cDNAs isolated from liver and kidney were shown to be identical, consisting of a 1008 bp open reading frame (ORF) encoding 336 amino acid residues with a molecular mass of 38091 Da. The rat ACMSD ORF was inserted into a mammalian expression vector, before transfection into human hepatoma HepG2 cells. The transfected cells expressed ACMSD activity, whereas the enzyme activity was not detected in uninfected parental HepG2 cells. The distribution of ACMSD mRNA expression in various tissues was investigated in the rat by RT-PCR. ACMSD was expressed in the liver and kidney, but not in the other principal organs examined.

L-2,4-diaminobutyric acid decarboxylase activity responsible for the formation of 1,3-diaminopropane in Enterobacter aerogenes.

High content of 1,3-diaminopropane (DAP), a normally minor derivative of polyamine metabolism, have been observed in cells of Enterobacter aerogenes. Supplementation of the growth medium with L-2,4-diaminobutyric acid (L-DABA) resulted in increased production of DAP, but not if supplemented with spermidine. On the basis of these observations, the biosynthetic route for DAP was evaluated. It has appeared that this bacterium possesses a novel enzyme activity catalysing the formation of DAP from L-DABA. Lack of the activity for oxidative cleavage of spermidine yielding DAP suggests that the enzyme termed DABA decarboxylase is responsible for the formation of DAP in this bacterium. The enzyme was partially purified 360-fold and some properties were examined. The pH optimum for the activity was 7.75-8.0, and the enzyme showed an absolute requirement for pyridoxal 5'-phosphate with the Km value of 41 microM. The Km value for L-DABA was 0.32 mM, and neither L-2,3-diaminopropionic acid, L-ornithine nor L-lysine showed detectable substrate activity towards the partially purified enzyme. Mg2+ and dithiothreitol greatly activated the enzyme.

Phosphoenolpyruvate carboxylase in lupin nodules and roots. Identification of the enzymatic forms.

Phosphoenolpyruvate carboxylase (PEPC) EC 4.1.1.31 was extracted from nodules and roots of 2-day-old seedlings of lupin (Lupinus luteus L.). Chromatography on DEAE-cellulose of the nodule extract gave two forms of the enzyme: PEPC I and PEPC II eluted at 0.3-0.35 M and 0.41-0.53 M Tris buffer, respectively. A third form PEPC III from lupin roots was eluted from DEAE-cellulose column at the same buffer concentration as PEPC II from nodules. PEPC I and PEPC II eluted at 0.3-0.35 M and 0.41-0.53 M Tris buffer, more active in the 6-week-old nodules binding effectively nitrogen than in the 12-week-old ones.

Role of pyruvate and S-adenosylmethioine in activating the pyruvate formate-lyase of Escherichia coli.

The pyruvate formate-lyase activity of extracts of Escherichia coli is stimulated and the dilution effect is abolished by the addition of pyruvate to the extract. The activity can be purified fourfold from pyruvate-supplemented extracts by isoelectric precipitation under anaerobic conditions. The activity of extracts not supplemented with pyruvate has been separated into two fractions by treatment with protamine sulfate-fraction PS, the soluble portion, and fraction N, an extract of the precipitate formed upon the addition of protamine sulfate. After treatment of these fractions with charcoal, pyruvate formate-lyase activity is stimulated by the addition of S-adenosylmethionine. When sodium pyruvate is added to the crude extract before the fractionation, fraction PS has full enzymatic activity and is not stimulated by fraction N or by S-adenosylmethionine. Incubation of the inactive fractions with pyruvate and S-adenosylmethionine in the absence of other substrates similarly results in a highly active preparation, not subject to the "dilution effect" obtained when the fractions are added separately to the assay. These observations suggest that the component in the protamine supernatant fraction is activated by the other fraction and that S-adenosylmethionine and pyruvate are required for the activation reaction. The activating factor present in the protamine precipitate fraction may be further purified by heating for 10 min at 100 C under H(2) atmosphere. The yield of this factor from crude extract is not affected by activation of the pyruvate formate-lyase of the extract, indicating that the factor acts catalytically. The requirement for pyruvate is only partially satisfied by alpha-ketobutyrate and not at all by other alpha-keto acids, acetyl phosphate, or adenosine triphosphate. The rate of activation is maximal at 0.01 m sodium pyruvate and 3 x 10(-4)mS-adenosylmethionine; it is linearly dependent on the amount of activating factor added. The rate of activation is the same when the activation reaction is initiated by addition of any of the four required components, indicating that no slow step of activation can be carried out by any three of the components. A similar pyruvate formate-lyase system was found in extracts of the methionine/B(12) autotroph 113-3, grown with methionine supplement, indicating that vitamin B(12) derivatives do not participate in the system.