Degradation pathway of the phosphonate ciliatine: crystal structure of 2-aminoethylphosphonate transaminase

Phosphonates allow certain organisms to thrive in otherwise hostile environments, and 2-aminoethylphosphonate (AEP) is a precursor of many cellular phosphonates. AEP transaminase (AEPT) is an enzyme essential to phosphonate synthesis and degradation pathways. The crystal structure of AEP transaminase was determined by multiwavelength anomalous diffraction of 66 selenium atoms. The refined structure at 2.2 A resolution revealed an overall fold and active site location similar to those of the dimeric, two-domain structure of type I aminotransferases. The active site contains a cofactor, pyridoxal 5'-phosphate (PLP), and the product phosphonoacetaldehyde. Comparison with other type I aminotransferase structures shows that the PLP-protein interactions are conserved. Modeling of bound substrates and products reveals the structural basis for AEP recognition and the stereospecificity of proton elimination at the alpha-carbon and indicates conformational changes along the reaction pathway.     

Purification and properties of 2-aminoethylphosphonate:pyruvate aminotransferase from Pseudomonas aeruginosa

2-Aminoethylphosphonate aminotransferase has been purified to homogeneity with a yield of 15% from cell extracts of Pseudomonas aeruginosa. The molecular weight of the enzyme was estimated by gel filtration to be 65000 +/- 2000. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis yielded a molecular weight of 16500 +/- 1000, suggesting a tetrameric model for this protein. The absorption spectrum exhibits maxima at 280 nm, 335 nm and 415 nm which are characteristic of a pyridoxal-phosphate-dependent enzyme: 4 mol of pyridoxal 5'-phosphate/mol of enzyme have been found. This aminotransferase catalyzes the transfer of the amino group of 2-aminoethylphosphonate (ciliatine) to pyruvate to give 2-phosphonoacetaldehyde and alanine. A pH optimum between 8.5-9 and an activity increasing from 30 degrees C to 50 degrees C have been observed. The reaction follows Michaelis-Menten kinetics with Km values of 3.85 mM and 3.5 mM for ciliatine and pyruvate respectively. This enzyme shows a very high specificity since ciliatine and pyruvate are the only amino donor and acceptor respectively. Methyl, ethyl and propylphosphonic acids are better competitors towards ciliatine than their alpha-amino derivatives. 3-Aminopropylphosphonate, the higher homologue of ciliatine, is recognized neither as a substrate nor as an inhibitor. The enzyme activity is significantly affected by carbonyl reagents and by HgCl2. Transamination of 2-aminoethylphosphonate is the first step of a double-step pathway which leads to the cleavage of its C-P bond.     

Characterization of an arginine:pyruvate transaminase in arginine catabolism of Pseudomonas aeruginosa PAO1

The arginine transaminase (ATA) pathway represents one of the multiple pathways for L-arginine catabolism in Pseudomonas aeruginosa. The AruH protein was proposed to catalyze the first step in the ATA pathway, converting the substrates L-arginine and pyruvate into 2-ketoarginine and L-alanine. Here we report the initial biochemical characterization of this enzyme. The aruH gene was overexpressed in Escherichia coli, and its product was purified to homogeneity. High-performance liquid chromatography and mass spectrometry (MS) analyses were employed to detect the presence of the transamination products 2-ketoarginine and L-alanine, thus demonstrating the proposed biochemical reaction catalyzed by AruH. The enzymatic properties and kinetic parameters of dimeric recombinant AruH were determined by a coupled reaction with NAD(+) and L-alanine dehydrogenase. The optimal activity of AruH was found at pH 9.0, and it has a novel substrate specificity with an order of preference of Arg > Lys > Met > Leu > Orn > Gln. With L-arginine and pyruvate as the substrates, Lineweaver-Burk plots of the data revealed a series of parallel lines characteristic of a ping-pong kinetic mechanism with calculated V(max) and k(cat) values of 54.6 +/- 2.5 micrromol/min/mg and 38.6 +/- 1.8 s(-1). The apparent K(m) and catalytic efficiency (k(cat)/K(m)) were 1.6 +/- 0.1 mM and 24.1 mM(-1) s(-1) for pyruvate and 13.9 +/- 0.8 mM and 2.8 mM(-1) s(-1) for l-arginine. When L-lysine was used as the substrate, MS analysis suggested Delta(1)-piperideine-2-carboxylate as its transamination product. These results implied that AruH may have a broader physiological function in amino acid catabolism.     

Rhodobacter capsulatus 1-deoxy-D-xylulose 5-phosphate synthase: steady-state kinetics and substrate binding

1-Deoxy-d-xylulose 5-phosphate synthase (DXP synthase) catalyzes the thiamine diphosphate (TPP)-dependent condensation of pyruvate and d-glyceraldehyde 3-phosphate (GAP) to yield DXP in the first step of the methylerythritol phosphate pathway for isoprenoid biosynthesis. Steady-state kinetic constants for DXP synthase calculated from the initial velocities measured at varying concentrations of substrates were as follows: k(cat) = 1.9 +/- 0.1 s(-1), K(m)(GAP) = 0.068 +/- 0.001 mM, and K(m)(pyruvate) = 0.44 +/- 0.05 mM for pyruvate and GAP; k(cat) = 1.7 +/- 0.1 s(-1), K(m)(d-glyceraldehyde) = 33 +/- 3 mM, and K(m)(pyruvate) = 1.9 +/- 0.5 mM for d-glyceraldehyde and pyruvate. beta-Fluoropyruvate was investigated as a dead-end inhibitor for pyruvate. Double-reciprocal plots showed a competitive inhibition pattern with respect to pyruvate and noncompetitive inhibition with respect to GAP/d-glyceraldehyde. (14)CO(2) trapping experiments demonstrated that the binding of both substrates (pyruvate and GAP/d-glyceraldehyde) is required for the formation of a catalytically competent enzyme-substrate complex. These results are consistent with an ordered mechanism for DXP synthase where pyruvate binds before GAP/d-glyceraldehyde.     

Characterization of a bifunctional archaeal acyl coenzyme A carboxylase

Acyl coenzyme A carboxylase (acyl-CoA carboxylase) was purified from Acidianus brierleyi. The purified enzyme showed a unique subunit structure (three subunits with apparent molecular masses of 62, 59, and 20 kDa) and a molecular mass of approximately 540 kDa, indicating an alpha(4)beta(4)gamma(4) subunit structure. The optimum temperature for the enzyme was 60 to 70 degrees C, and the optimum pH was around 6.4 to 6.9. Interestingly, the purified enzyme also had propionyl-CoA carboxylase activity. The apparent K(m) for acetyl-CoA was 0.17 +/- 0.03 mM, with a V(max) of 43.3 +/- 2.8 U mg(-1), and the K(m) for propionyl-CoA was 0.10 +/- 0.008 mM, with a V(max) of 40.8 +/- 1.0 U mg(-1). This result showed that A. brierleyi acyl-CoA carboxylase is a bifunctional enzyme in the modified 3-hydroxypropionate cycle. Both enzymatic activities were inhibited by malonyl-CoA, methymalonyl-CoA, succinyl-CoA, or CoA but not by palmitoyl-CoA. The gene encoding acyl-CoA carboxylase was cloned and characterized. Homology searches of the deduced amino acid sequences of the 62-, 59-, and 20-kDa subunits indicated the presence of functional domains for carboxyltransferase, biotin carboxylase, and biotin carboxyl carrier protein, respectively. Amino acid sequence alignment of acetyl-CoA carboxylases revealed that archaeal acyl-CoA carboxylases are closer to those of Bacteria than to those of Eucarya. The substrate-binding motifs of the enzymes are highly conserved among the three domains. The ATP-binding residues were found in the biotin carboxylase subunit, whereas the conserved biotin-binding site was located on the biotin carboxyl carrier protein. The acyl-CoA-binding site and the carboxybiotin-binding site were found in the carboxyltransferase subunit.     

Investigation of the substrate binding and catalytic groups of the P-C bond cleaving enzyme, phosphonoacetaldehyde hydrolase.

Kinetic studies with substrate analogs and group-directed chemical modification agents were carried out for the purpose of identifying the enzyme-substrate interactions required for phosphonoacetaldehyde (P-Ald) binding and catalyzed hydrolysis by P-Ald hydrolase (phosphonatase). Malonic semialdehyde (Ki = 1.6 mM), phosphonoacetate (Ki = 10 mM), phosphonoethanol (Ki = 10 mM), and fluorophosphate (Ki = 20 mM) were found to be competitive inhibitors of the enzyme but not substrates. Thiophosphonoacetaldehyde and acetonyl phosphonate underwent phosphonatase-catalyzed hydrolysis but at 20-fold and 140-fold slower rates, respectively, than did P-Ald. In the presence of NaBH4, acetonyl-phosphonate inactivated phosphonatase at a rate exceeding that of its turnover. Sequence analysis of the radiolabeled tryptic peptide generated from [3-3H]acetonylphosphonate/NaBH4-treated phosphonatase revealed that Schiff base formation had occurred with the catalytic lysine. From the Vm/Km and Vm pH profiles for phosphonatase-catalyzed P-Ald hydrolysis, an optimal pH range of 6-8 was defined for substrate binding and catalysis. The pH dependence of inactivation by acetylation of the active site lysine with acetic anhydride and 2,4-dinitrophenyl acetate evidenced protonation of the active site lysine residue as the cause for activity loss below pH 6. The pH dependence of inactivation of an active site cysteine residue with methyl methanethiol-sulfonate indicated that deprotonation of this residue may be the cause for the loss of enzyme activity above pH 8.     

Geranylgeranylglyceryl phosphate synthase. Characterization of the recombinant enzyme from Methanobacterium thermoautotrophicum.

Geranylgeranylglyceryl diphosphate synthase (GGGP synthase) catalyzes alkylation of (S)-glyceryl phosphate [(S)-GP] by geranylgeranyl diphosphate (GGPP) to produce (S)-geranylgeranylglyceryl phosphate [(S)-GGGP]. This reaction is the first committed step in the biosynthesis of ether-linked membrane lipids in Archaea. The gene encoding GGGP synthase from Methanobacterium thermoautotrophicum was cloned using probes designed from the N-terminal sequence determined from the purified enzyme. The open reading frame, which encoded a protein of 245 amino acids, was inserted into a pET expression vector and expressed in Escherichia coli. The recombinant GGGP synthase was purified to homogeneity. The enzyme is active as a homopentamer, as determined by size exclusion chromatography and equilibrium sedimentation experiments. GGGP synthase has optimal activity at 55 degrees C in pH 8.0 buffer containing 1 mM MgCl(2). V(max) = 4.0 +/- 0.1 micromol min(-1) mg(-1) (k(cat) = 0.34 +/- 0.03 s(-1) for pentameric GGGP synthase assuming all subunits are fully active), K(m)((S)-GP) = 13.5 +/- 1.0 microM, and K(m)(GGPP) = 506 +/- 47 nM. These steady-state catalytic constants were identical to those for enzyme isolated from cell extracts of M. thermoautotrophicum [Chen, A., Zhang, D., and Poulter, C. D. (1993) J. Biol. Chem. 268, 21701-21705]. Alignment of seven putative archaeal GGGP synthase sequences revealed a number of highly conserved residues consisting of five aspartate/glutamates, three serine/threonines, two prolines, and five glycines, including a conserved GGG motif.     

omega-Amino acid:pyruvate transaminase from Alcaligenes denitrificans Y2k-2: a new catalyst for kinetic resolution of beta-amino acids and amines

Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed omega-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic beta-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for L-beta-amino-n-butyric acid (L-beta-ABA). The enzyme converts various beta-amino acids and amines to the corresponding beta-keto acids and ketones by using pyruvate as an amine acceptor. The apparent K(m) and V(max) for L-beta-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM L-beta-ABA, the apparent K(m) and V(max) for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM D,L-beta-ABA, producing optically pure D-beta-ABA (99% enantiomeric excess) with 53% conversion.     

Purification,characterization, and molecular cloning of a novel amine:pyruvate transaminase from Vibrio fluvialis JS17

A transaminase from Vibrio fluvialis JS17 showing activity toward chiral amines was purified to homogeneity and its enzymatic properties were characterized. The transaminase showed an apparent molecular mass of 100 kDa as determined by gel filtration chromatography and a subunit mass of 50 kDa by MALDI-TOF mass spectrometry, suggesting a dimeric structure. The enzyme had an isoelectric point of 5.4 and its absorption spectrum exhibited maxima at 320 and 405 nm. The optimal pH and temperature for enzyme activity were 9.2 and 37 degrees C, respectively. Pyruvate and pyridoxal 5'-phosphate increased enzyme stability whereas (S)-alpha-methylbenzylamine reversibly inactivated the enzyme. The transaminase gene was cloned from a V. fluvialis JS17 genomic library. The deduced amino acid sequence (453 residues) showed significant homology with omega-amino acid:pyruvate transaminases (omega-APT) from various bacterial strains (80 identical residues with four omega-APTs). However, of 159 conserved residues in the four omega-APTs, 79 were not conserved in the transaminase from V. fluvialis JS17. Taken together with the sequence homology results, and the lack of activity toward beta-alanine (a typical amino donor for the omega-APT), the results suggest that the transaminase is a novel amine:pyruvate transaminase that has not been reported to date.     

Pyruvate dehydrogenase kinase isoform 2 activity limited and further inhibited by slowing down the rate of dissociation of ADP

Pyruvate dehydrogenase kinase 2 (PDK2) activity is enhanced by the dihydrolipoyl acetyltransferase core (E2 60mer) that binds PDK2 and a large number of its pyruvate dehydrogenase (E1) substrate. With E2-activated PDK2, K(+) at approximately 90 mM and Cl(-) at approximately 60 mM decreased the K(m) of PDK2 for ATP and competitive K(i) for ADP by approximately 3-fold and enhanced pyruvate inhibition. Comparing PDK2 catalysis +/- E2, E2 increased the K(m) of PDK2 for ATP by nearly 8-fold (from 5 to 39 microM), increased k(cat) by approximately 4-fold, and decreased the requirement for E1 by at least 400-fold. ATP binding, measured by a cold-trapping technique, occurred at two active sites with a K(d) of 5 microM, which equals the K(m) and K(d) of PDK2 for ATP measured in the absence of E2. During E2-aided catalysis, PDK2 had approximately 3 times more ADP than ATP bound at its active site, and the pyruvate analogue, dichloroacetate, led to 16-fold more ADP than ATP being bound (no added ADP). Pyruvate functioned as an uncompetitive inhibitor versus ATP, and inclusion of ADP transformed pyruvate inhibition to noncompetitive. At high pyruvate levels, pyruvate was a partial inhibitor but also induced substrate inhibition at high ATP levels. Our results indicate that, at physiological salt levels, ADP dissociation is a limiting step in E2-activated PDK2 catalysis, that PDK2.[ADP or ATP].pyruvate complexes form, and that PDK2.ATP.pyruvate.E1 reacts with PDK2.ADP.pyruvate accumulating.     

Synthesis of enantiomerically pure trans-(1R,2R)- and cis-(1S,2R)-1-amino-2-indanol by lipase and omega-transaminase

Syntheses of trans-(1R,2R) and cis-(1S,2R)-1-amino-2-indanol (AI) were accomplished by a series of enantioselective enzymatic reactions using lipase and transaminase (TA). Lipase catalysed enantioselective hydrolysis of 2-acetoxyindanone was employed to prepare (R)-2-hydroxy indanone (HI). trans-AI (5 mM) (de > 98%) was produced from 20 mM (R)-2- HI using omega-TA and 50 mM (S)-1-aminoindan as an amino donor in water-saturated ethyl acetate. For the production of cis-AI, the diastereomeric (2R)-AI was synthesized from (R)-2-HI using reductive amination, and the kinetic resolution was performed with omega-TA. The enantioselectivity of omega-TA for (2R)-AI was increased to 22.1 in the presence of 5% gamma-cyclodextrin. cis-AI (15.4 mM) (96% de) was obtained from 40 mM (2R)-AI using 30 mM pyruvate and omega-TA (25 mg) in 10 mL of 100 mM phosphate buffer (pH 7.0).     

Treponema denticola cystalysin catalyzes beta-desulfination of L-cysteine sulfinic acid and beta-decarboxylation of L-aspartate and oxalacetate

Pyridoxal 5'-phosphate-dependent cystalysin from Treponema denticola catalyzes the beta-displacement of the beta-substituent from both L-aspartate and L-cysteine sulfinic acid. The steady-state kinetic parameters for beta-desulfination of L-cysteine sulfinic acid, k(cat) and K(m), are 89+/-7 s(-1) and 49+/-9 mM, respectively, whereas those for beta-decarboxylation of L-aspartate are 0.8+/-0.1 s(-1) and 280+/-70 mM. Moreover, cystalysin in the pyridoxamine 5'-phosphate form has also been found to catalyze beta-decarboxylation of oxalacetate as shown by consumption of oxalacetate and a concomitant production of pyruvate. The k(cat) and K(m) of this reaction are 0.15+/-0.01 s(-1) and 13+/-2 mM, respectively. Possible mechanistic and physiological implications are discussed.     

The vnd/NK-2 homeodomain: thermodynamics of reversible unfolding and DNA binding for wild-type and with residue replacements H52R and H52R/T56W in helix III

The conformational stabilities of the vnd (ventral nervous system defective)/NK-2 homeodomain [HD(wt); residues 1-80 that encompass the 60-residue homeodomain] and those harboring mutations in helix III of the DNA recognition site [HD(H52R) and HD(H52R/T56W)] have been investigated by differential scanning calorimetry (DSC) and ellipticity changes at 222 nm. Thermal unfolding reactions at pH 7.4 are reversible and repeatable in the presence of 50-500 mM NaCl with DeltaC(p) = 0.52 +/- 0.04 kcal K(-1) mol(-1). A substantial stabilization of HD(wt) is produced by 50 mM phosphate or by the addition of 100-500 mM NaCl to 50 mM Hepes, pH 7.4, buffer (from T(m) = 35.5 degrees C to T(m) 43-51 degrees C; DeltaH(vH) congruent with 47 +/- 5 kcal mol(-1)). The order of stability is HD(H52R/T56W) > HD(H52R) > HD(wt), irrespective of the anions present. Progress curves for ellipticity changes at 222 nm as a function of increasing temperature are fitted well by a two-state unfolding model, and the cooperativity of secondary structure changes is greater for mutant homeodomains than for HD(wt) and also is increased by adding 100 mM NaCl to Hepes buffer. A 33% quench of the intrinsic tryptophanyl residue fluorescence of HD(wt) by phosphate binding (K(D)' = 2.6 +/- 0.3 mM phosphate) is reversed approximately 60% by DNA binding. Thermodynamic parameters for vnd/NK-2 homeodomain proteins binding sequence-specific 18 bp DNA have been determined by isothermal titration calorimetry (10-30 degrees C). Values of DeltaC(p) are +0.25, -0.17, and -0.10 +/- 0.04 kcal K(-1) mol(-1) for HD(wt), HD(H52R), and HD(H52R/T56W) binding duplex DNA, respectively. Interactions of homeodomains with DNA are enthalpically controlled at 298 K and pH 7.4 with corresponding DeltaH values of -6.6 +/- 0.5, -10.8 +/- 0.1, and -9.0 +/- 0.6 kcal mol(-1) and DeltaG' values of -11.0 +/- 0.1, -11.0 +/- 0.1, and -11.3 +/- 0.3 kcal mol(-1) with a binding stoichiometry of 1.0 +/- 0.1. Thermodynamic parameters for DNA binding are not predicted from homeodomain structural changes that occur upon complexing to DNA and must reflect also solvent and possibly DNA rearrangements.     

31P NMR probes of sipunculan erythrocytes containing the O2-carrying protein hemerythrin

Reported are the first examinations by 31P NMR of erythrocytes containing the non-heme iron O2-carrying protein hemerythrin (Hr). Intact coelomic erythrocytes from the sipunculids Phascolopsis gouldii and Themiste zostericola were shown by 31P NMR to contain O-phosphorylethanolamine and 2-aminoethylphosphonate as the major soluble phosphorus metabolites. This combination of major metabolites appears to be unique to sipunculan erythrocytes. Nucleoside triphosphates and mannose 1-phosphate were present in lower concentrations. The concentration of O-phosphorylethanolamine within P. gouldii erythrocytes was established to be greater than 20 mM. T. zostericola erythrocytes contained relatively high levels of 2-aminoethylphosphonate (on the order of 0.1 M) and lower levels of O-phosphorylethanolamine compared with those of P. gouldii. For P. gouldii and T. zostericola the intracellular pHs were determined to be 7.2 +/- 0.1 and 7.1 +/- 0.1, respectively, in air-equilibrated erythrocytes, and 6.5 +/- 0.1 in anaerobic P. gouldii erythrocytes. O-Phosphorylethanolamine was found to bind weakly to P. gouldii metHr (Kf approximately 7 M-1). This interaction is best characterized by either negative cooperativity or nonspecific binding. O-Phosphorylethanolamine strongly inhibits azide binding to the iron site of P. gouldii metHr at pH 7.2. The rate of azide binding decreases by approximately 85-fold in the presence of 0.33 M O-phosphorylethanolamine. However, neither O-phosphorylethanolamine nor 2-aminoethylphosphonate at 0.33 M was found to have any significant effect on O2 affinity of P. gouldii deoxyHr. Alternative functions for the two metabolites are suggested.     

Subcellular distribution and properties of kynurenine pyruvate transaminase in rat kidney.

Kynurenine transaminase activity in rat kidney was found in both the mitochondrial and supernatant fractions. These fractions contained (a) kynurenine pyruvate transaminase, which showed a preference for pyruvate as amino acceptor, and had a pH optimum between 8.0 and 8.5, and (b) kynurenine 2-oxoglutarate transaminase, with a preference for 2-oxoglutarate and a pH optimum between 6.0 and 6.5. The apparent Km value of the former enzyme for L-kynurenine was much lower than that of the latter enzyme.     

The pH dependence of the reductive carboxylation of pyruvate by malic enzyme

The maximum velocity of the malic enzyme (L-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) reductive carboxylation of pyruvate and V/KCO2 are pH-independent from pH 5.5 to pH 8.5. V/K for pyruvate exhibits pK values values of 6.50 +/- 0.25 and 7.25 +/- 0.25. These data suggest that the binding of pyruvate locks the protonation state of enzyme. In addition, the pK values are within experimental error identical for the pH dependence of V/Kmalate and V/Kpyruvate. Thus, the catalytic groups appear to have reverse protonation states in the two reaction directions. The ratio of (V/Kmalate)/(V/Kpyruvate) is 100, suggesting that the protonation state of enzyme is optimum in the malate oxidative decarboxylation direction. Thus, the group with a pK of about 6 is unprotonated and the group with a pK of 7.5 is protonated for malate decarboxylation, and the opposite is true for pyruvate reductive carboxylation.     

Functional and structural interactions of the transmembrane domain X of NhaA, Na+/H+ antiporter of Escherichia coli, at physiological pH

The 3D structure of Escherichia coli NhaA, determined at pH 4, provided the first structural insights into the mechanism of antiport and pH regulation of a Na+/H+ antiporter. However, because NhaA is activated at physiological pH (pH 7.0-8.5), many questions pertaining to the active state of NhaA have remained open, including the physiological role of helix X. Using a structural-based evolutionary approach in silico, we identified a segment of most conserved residues in the middle of helix X. These residues were then used as targets for functional studies at physiological pH. Cysteine-scanning mutagenesis showed that Gly303, in the middle of the conserved segment, is an essential residue and Cys replacement of Lys300 retains only Li+/H+ antiporter activity, with a 20-fold increase in the apparent KM for Li+. Cys replacements of Leu296 and Gly299 increase the apparent KM of the Na+/H+ antiporter for both Na+ and Li+. Accessibility test to N-ethylmaleimide and 2-sulfonatoethyl methanethiosulfonate showed that G299C, K300C, and G303C are accessible to the cytoplasm. Suppressor mutations and site-directed chemical cross-linking identified a functional and/or structural interaction between helix X (G295C) and helix IVp (A130C). While these results were in accordance with the acid-locked crystal structure, surprisingly, conflicting data were also obtained; E78C of helix II cross-links very efficiently with several Cys replacements of helix X, and E78K/K300E is a suppressor mutation of K300E. These results reveal that, at alkaline pH, the distance between the conserved center of helix X and E78 of helix II is drastically decreased, implying a pH-induced conformational change of one or both helices.     

4-Aminobutyrate:2-oxoglutarate aminotransferase of Streptomyces griseus: purification and properties

4-Aminobutyrate: 2-oxoglutarate aminotransferase of Streptomyces griseus was purified to homogeneity on disc electrophoresis. The relative molecular mass of the enzyme was found to be 100 000 +/- 10 000 by a gel filtration method. The enzyme consists of two subunits identical in molecular mass (Mr 50 000 +/- 1000). The transaminase is composed of 486 amino acids/subunit containing 10 and 12 residues of half-cystine and methionine respectively. The NH2-terminal amino acid sequence of the enzyme was determined to be Thr-Ala-Phe-Pro-Gln. The enzyme exhibits absorption maxima at 278 nm, 340 nm and 415 nm with a molar absorption coefficient of 104 000, 11 400 and 7280 M-1 cm-1 respectively. The pyridoxal 5'-phosphate content was calculated to be 2 mol/mol enzyme. The enzyme has a maximum activity in the pH range of 7.5-8.5 and at 50 degrees C. The enzyme is stable at pH 6.0-10.0 and at temperatures up to 50 degrees C. Pyridoxal 5'-phosphate protects the enzyme from thermal inactivation. The enzyme catalyzes the transamination of omega-amino acids with 2-oxoglutarate; 4-aminobutyrate is the best amino donor. The Michaelis constants are 3.3 mM for 4-aminobutyrate and 8.3 mM for 2-oxoglutarate. Low activity was observed with beta-alanine. In addition to omega-amino acids the enzyme catalyzes transamination with ornithine and lysine; in both cases the D isomer is preferred. Carbonyl reagents and sulfhydryl reagents inhibit the enzyme activity. Chelating agents, non-substrate L and D-2-amino acids, and metal ions except cupric ion showed no effect on the enzyme activity.     

Streptomyces beta-alanine:alpha-ketoglutarate aminotransferase, a novel omega-amino acid transaminase. Purification, crystallization, and enzymologic properties

An enzyme which catalyzes the transamination of beta-alanine with alpha-ketoglutarate was purified to homogeneity from Streptomyces griseus IFO 3102 and crystallized. Molecular weight of the enzyme was found to be 185,000 +/- 10,000 by a gel-filtration method. The enzyme consists of four subunits identical in molecular weight (51,000 +/- 1,000). The transaminase is composed of 483 amino acids/subunit containing 7 and 8 residues of half-cystine and methionine, respectively. The enzyme exhibits absorption maxima at 278 and 415 nm. The pyridoxal 5'-phosphate content was determined to be 4 mol/mol of enzyme. The enzyme catalyzes transamination of omega-amino acids including taurine and hypotaurine. beta-Alanine and DL-beta-aminoisobutyrate served as a good amino donor; the Michaelis constants are 8.0 and 12.5 mM, respectively. alpha-Ketoglutarate is the only amino acceptor (Km = 4.0 mM); pyruvate and oxalacetate are inactive. Based on the substrate specificity, the terminology of beta-alanine:alpha-ketoglutarate transaminase is proposed for the enzyme. Carbonyl reagents, HgCl2,DL-gabaculine, and alpha-fluoro-beta-alanine strongly inhibited the enzyme.     

Electricity production from xylose in fed-batch and continuous-flow microbial fuel cells

A medium-scale (0.77 l) air-cathode, brush-anode microbial fuel cell (MFC) operated in fed-batch mode using xylose (20 mM) generated a maximum power density of 13 +/- 1 W/m(3) (673 +/- 43 mW/m(2)). Xylose was rapidly removed (83.5%) within 8 h of a 60-h cycle, with 42.1% of electrons in intermediates (8.5 +/- 0.2 mM acetate, 5.9 +/- 0.01 mM ethanol, 4.3 +/- 0.1 mM formate, and 1.3 +/- 0.03 mM propionate), 9.1% captured as electricity, 16.1% in the remaining xylose, and 32.7% lost to cell storage, biomass, and other processes. The final Coulombic efficiency was 50%. At a higher initial xylose concentration (54 mM), xylose was again rapidly removed (86.9% within 24 h of a 116-h cycle), intermediates increased in concentration (18.4 +/- 0.4 mM acetate, 7.8 +/- 0.4 mM ethanol and 2.1 +/- 0.2 mM propionate), but power was lower (5.2 +/- 0.4 W/m(3)). Power was increased by operating the reactor in continuous flow mode at a hydraulic retention time of 20 h (20 +/- 1 W/m(3)), with 66 +/- 1% chemical oxygen demand removal. These results demonstrate that electricity generation is sustained over a cycle primarily by stored substrate and intermediates formed by fermentation and that the intermediates produced vary with xylose loading.