Characterization of the 4-hydroxybenzoyl-coenzyme A thioesterase from Arthrobacter sp. strain SU

The Arthrobacter sp. strain SU 4-chlorobenzoate (4-CBA) dehalogenation pathway converts 4-CBA to 4-hydroxybenzoate (4-HBA). The pathway operon contains the genes fcbA, fcbB, and fcbC (A. Schmitz, K. H. Gartemann, J. Fiedler, E. Grund, and R. Eichenlaub, Appl. Environ. Microbiol. 58:4068-4071, 1992). Genes fcbA and fcbB encode 4-CBA-coenzyme A (CoA) ligase and 4-CBA-CoA dehalogenase, respectively, whereas the function of fcbC is not known. We subcloned fcbC and expressed it in Escherichia coli, and we purified and characterized the FcbC protein. A substrate activity screen identified benzoyl-CoA thioesters as the most active substrates. Catalysis of 4-HBA-CoA hydrolysis to 4-HBA and CoA occurred with a k(cat) of 6.7 s(-1) and a K(m) of 1.2 micro M. The k(cat) pH rate profile for 4-HBA-CoA hydrolysis indicated optimal activity over a pH range of 6 to 10. The amino acid sequence of the FcbC protein was compared to other sequences contained in the protein sequence data banks. A large number of sequence homologues of unknown function were identified. On the other hand, the 4-HBA-CoA thioesterases isolated from 4-CBA-degrading Pseudomonas strains did not share significant sequence identity with the FcbC protein, indicating early divergence of the thioesterase-encoding genes.     

Investigation of the catalytic mechanism of the hotdog-fold enzyme superfamily Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-CoA thioesterase

The 4-hydroxybenzoyl-CoA (4-HB-CoA) thioesterase from Pseudomonas sp. strain CBS3 catalyzes the final step of the 4-chlorobenzoate degradation pathway, which is the hydrolysis of 4-HB-CoA to coenzyme A (CoA) and 4-hydroxybenzoate (4-HB). In previous work, X-ray structural analysis of the substrate-bound thioesterase provided evidence of the role of an active site Asp17 in nucleophilic catalysis [Thoden, J. B., Holden, H. M., Zhuang, Z., and Dunaway-Mariano, D. (2002) X-ray crystallographic analyses of inhibitor and substrate complexes of wild-type and mutant 4-hydroxybenzoyl-CoA thioesterase. J. Biol. Chem. 277, 27468-27476]. In the study presented here, kinetic techniques were used to test the catalytic mechanism that was suggested by the X-ray structural data. The time course for the multiple-turnover reaction of 50 μM [(14)C]-4-HB-CoA catalyzed by 10 μM thioesterase supported a two-step pathway in which the second step is rate-limiting. Steady-state product inhibition studies revealed that binding of CoA (K(is) = 250 ± 70 μM; K(ii) = 900 ± 300 μM) and 4-HB (K(is) = 1.2 ± 0.2 mM) is weak, suggesting that product release is not rate-limiting. A substantial D(2)O solvent kinetic isotope effect (3.8) on the steady-state k(cat) value (18 s(-1)) provided evidence that a chemical step involving proton transfer is the rate-limiting step. Taken together, the kinetic results support a two-chemical pathway. The microscopic rate constants governing the formation and consumption of the putative aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate were determined by simulation-based fitting of a kinetic model to time courses for the substrate binding reaction (5.0 μM 4-HB-CoA and 0.54 μM thioesterase), single-turnover reaction (5 μM [(14)C]-4-HB-CoA catalyzed by 50 μM thioesterase), steady-state reaction (5.2 μM 4-HB-CoA catalyzed by 0.003 μM thioesterase), and transient-state multiple-turnover reaction (50 μM [(14)C]-4-HB-CoA catalyzed by 10 μM thioesterase). Together with the results obtained from solvent (18)O labeling experiments, the findings are interpreted as evidence of the formation of an aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate that undergoes rate-limiting hydrolytic cleavage at the hydroxybenzoyl carbonyl carbon atom.     

Kinetic,Raman, NMR,and site-directed mutagenesis studies of the Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-CoA thioesterase active site

4-Hydroxybenzoyl-coenzyme A (4-HBA-CoA) thioesterase catalyzes the hydrolysis of 4-HBA-CoA to 4-hydroxybenzoate and CoA. X-ray crystallographic analysis of the liganded enzyme has shown that the benzoyl thioester and pantetheine moieties of the substrate ligand are bound in a narrow crevice while the nucleotide moiety rests on the protein surface (Thoden, J. B., Holden, H. M., Zhuang, Z. and Dunaway-Mariano, D. (2002) X-ray Crystallographic Analyses of Inhibitor and Substrate Complexes of Wild-type and Mutant 4-Hydroxybenzoyl-CoA Thioesterase, J. Biol. Chem., in press). Asp17 is positioned in the crevice, close to the substrate thioester C=O, which in turn interacts with the positive pole of an alpha-helix macrodipole. In this paper we report the results from spectral, mutagenesis, and kinetic studies which show (1) that substrate activation involves restricted thioester C=O conformational freedom and a modest enhancement of C=O bond polarization, (2) that the nucleotide unit of the substrate is bound through interaction with the protein surface, and (3) that Asp17 contributes a rate factor of 10(4), consistent with its proposed role of general base or nucleophile.     

Cloning and sequence analysis of genes for dehalogenation of 4-chlorobenzoate from Arthrobacter sp. strain SU.

Strains of Arthrobacter catalyze a hydrolytic dehalogenation of 4-chlorobenzoate (4-CBA) to p-hydroxybenzoate. The reaction requires ATP and coenzyme A (CoA), indicating activation of the substrate via a thioester, like that reported for Pseudomonas sp. strain CBS3 (J. D. Scholten, K.-H. Chang, P. C. Babbit, H. Charest, M. Sylvestre, and D. Dunaway-Mariano, Science 253:182-185, 1991). The dehalogenase genes of Arthrobacter sp. strain SU were cloned and expressed in Escherichia coli. Analyses of deletions indicate that dehalogenation depends on three open reading frames (ORFs) which are organized in an operon. There is extensive sequence homology to corresponding gene products in Pseudomonas sp. strain CBS3, suggesting that ORF1 and ORF2 encode a 4-CBA-CoA-ligase and a 4-CBA-CoA dehalogenase, respectively. ORF3 possibly represents a thioesterase, although no homology to the enzyme from Pseudomonas sp. strain CBS3 exists.     

Cloning and sequence analysis of genes for dehalogenation of 4-chlorobenzoate from Arthrobacter sp. strain SU.

Strains of Arthrobacter catalyze a hydrolytic dehalogenation of 4-chlorobenzoate (4-CBA) to p-hydroxybenzoate. The reaction requires ATP and coenzyme A (CoA), indicating activation of the substrate via a thioester, like that reported for Pseudomonas sp. strain CBS3 (J. D. Scholten, K.-H. Chang, P. C. Babbit, H. Charest, M. Sylvestre, and D. Dunaway-Mariano, Science 253:182-185, 1991). The dehalogenase genes of Arthrobacter sp. strain SU were cloned and expressed in Escherichia coli. Analyses of deletions indicate that dehalogenation depends on three open reading frames (ORFs) which are organized in an operon. There is extensive sequence homology to corresponding gene products in Pseudomonas sp. strain CBS3, suggesting that ORF1 and ORF2 encode a 4-CBA-CoA-ligase and a 4-CBA-CoA dehalogenase, respectively. ORF3 possibly represents a thioesterase, although no homology to the enzyme from Pseudomonas sp. strain CBS3 exists.     

X-ray crystallographic analyses of inhibitor and substrate complexes of wild-type and mutant 4-hydroxybenzoyl-CoA thioesterase

The metabolic pathway by which 4-chlorobenzoate is degraded to 4-hydroxybenzoate in the soil-dwelling microbe Pseudomonas sp. strain CBS-3 consists of three enzymes including 4-hydroxybenzoyl-CoA thioesterase. The structure of the unbound form of this thioesterase has been shown to contain the so-called "hot dog" fold with a large helix packed against a five-stranded anti-parallel beta-sheet. To address the manner in which the enzyme accommodates the substrate within the active site, two inhibitors have been synthesized, namely 4-hydroxyphenacyl-CoA and 4-hydroxybenzyl-CoA. Here we describe the structural analyses of the enzyme complexed with these two inhibitors determined and refined to 1.5 and 1.8 A resolution, respectively. These studies indicate that only one protein side chain, Ser(91), participates directly in ligand binding. All of the other interactions between the protein and the inhibitors are mediated through backbone peptidic NH groups, carbonyl oxygens, and/or solvents. The structures of the enzyme-inhibitor complexes suggest that both a hydrogen bond and the positive end of a helix dipole moment serve to polarize the electrons away from the carbonyl carbon of the acyl group, thereby making it more susceptible to nucleophilic attack. Additionally, these studies demonstrate that the carboxylate group of Asp(17) is approximately 3.2 A from the carbonyl carbon of the acyl group. To address the role of Asp(17), the structure of the site-directed mutant protein D17N with bound substrate has also been determined. Taken together, these investigations suggest that the reaction mechanism may proceed through an acyl enzyme intermediate.     

Characterization of the 4-Hydroxybenzoyl-Coenzyme A Thioesterase from Arthrobacter sp. Strain SU

The Arthrobacter sp. strain SU 4-chlorobenzoate (4-CBA) dehalogenation pathway converts 4-CBA to 4-hydroxybenzoate (4-HBA). The pathway operon contains the genes fcbA, fcbB, and fcbC (A. Schmitz, K. H. Gartemann, J. Fiedler, E. Grund, and R. Eichenlaub, Appl. Environ. Microbiol. 58:4068-4071, 1992). Genes fcbA and fcbB encode 4-CBA-coenzyme A (CoA) ligase and 4-CBA-CoA dehalogenase, respectively, whereas the function of fcbC is not known. We subcloned fcbC and expressed it in Escherichia coli, and we purified and characterized the FcbC protein. A substrate activity screen identified benzoyl-CoA thioesters as the most active substrates. Catalysis of 4-HBA-CoA hydrolysis to 4-HBA and CoA occurred with a k_cat of 6.7 s⁻¹ and a K_m of 1.2 μM. The k_cat pH rate profile for 4-HBA-CoA hydrolysis indicated optimal activity over a pH range of 6 to 10. The amino acid sequence of the FcbC protein was compared to other sequences contained in the protein sequence data banks. A large number of sequence homologues of unknown function were identified. On the other hand, the 4-HBA-CoA thioesterases isolated from 4-CBA-degrading Pseudomonas strains did not share significant sequence identity with the FcbC protein, indicating early divergence of the thioesterase-encoding genes.     

A crystalline pigment produced from 2-hydroxypyridine by arthrobacter crystallopoietes n.sp.

Summary A bacterium was isolated from soil which utilizes 2-hydroxypyridine as sole source of carbon and nitrogen. When grown on solid medium with this substrate massive amounts of green rectangular crystals are deposited extracellularly in the colony mass. The pigment producing organism proved to be a hitherto undescribed species to which the name Arthrobacter crystallopoietes was applied. The pigment formed is characterized qualitatively by the following properties: it is an oxidation product of 2-hydroxypyridine probably still containing a six-membered heterocyclic ring; it exists as an anion with an intense blue color in neutral or slightly alkaline solution and as a metal salt in the deposited crystals; it precipitates from acid solution as a red water-insoluble free acid; it can be reversible oxidized and reduced, being colorless in the reduced form; and in solution it is spontaneously oxidized by air, the reaction being very rapid at alkalineph. The ultraviolet, visible and infrared spectra of the blue and red forms are presented. The properties of the pigment show that it is a member of a chemically poorly defined group of compounds termed azaquinones and that it is related to but not identical with pigments produced by the bacterial oxidation of nicotine, nicotinic acid and isonicotinic acid.This investigation was supported in part by grants G9882 and GB736 from the National Science Foundation.     

Enzymatic dehalogenation of 4-chlorobenzoate by extracts from Arthrobacter sp. SU DSM 20407

In extracts from Arthrobacter sp. SU DSM 20407 an enzyme was detectable, that converted 4-chlorobenzoate into 4-hydroxybenzoate. This conversion was also observed when no oxygen was present in the reaction mixture. Boiling for 5 min destroyed the enzyme activity. 4-Bromo- and 4-iodobenzoate were substrates for the enzyme too, but not 4-fluorobenzoate, 4-chlorophenylacetate and 4-chlorocinnamic acid. The enzyme showed optimum activity at 16 degrees C and at pH 7-7.5. The specific activity in the extracts varied between 0.5 and 5 mU/mg of protein. Zn2+ and Cu2+ inhibited the enzyme, while H2O2 slightly activated. In contrast to all other 4-chlorobenzoate dehalogenases described before the enzyme was not inhibited by EDTA, nor was it activated by Mn2+. Other divalent ions also had no effect. The molecular mass of the enzyme was 45,000 +/- 5,000 Da as judged by gel-filtration.     

The Catalytic Mechanism of the Hotdog-fold Enzyme Superfamily 4-Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp. Strain SU

The hotdog-fold enzyme 4-hydroxybenzoyl-coenzyme A (4-HB-CoA) thioesterase from Arthrobacter sp. strain AU catalyzes the hydrolysis of 4-HB-CoA to form 4-hydroxybenzoate (4-HB) and coenzyme A (CoA) in the final step of the 4-chlorobenzoate dehalogenation pathway. Guided by the published X-ray structures of the liganded enzyme (Thoden, J. B., Zhuang, Z., Dunaway-Mariano, D., and Holden H. M. (2003) J.Biol. Chem. 278, 43709-43716), a series of site-directed mutants were prepared for testing the roles of active site residues in substrate binding and catalysis. The mutant thioesterases were subjected to X-ray structure determination to confirm retention of the native fold, and in some cases, to reveal changes in the active site configuration. In parallel, the wild-type and mutant thioesterases were subjected to transient and steady-state kinetic analysis, and to (18)O-solvent labeling experiments. Evidence is provided that suggests that Glu73 functions in nucleophilic catalysis, that Gly65 and Gln58 contribute to transition-state stabilization via hydrogen bond formation with the thioester moiety and that Thr77 orients the water nucleophile for attack at the 4-hydroxybenzoyl carbon of the enzyme-anhydride intermediate. The replacement of Glu73 with Asp was shown to switch the function of the carboxylate residue from nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to a single-step hydrolysis reaction. The E73D/T77A double mutant regained most of the catalytic efficiency lost in the E73D single mutant. The results from (31)P NMR experiments indicate that the substrate nucleotide unit is bound to the enzyme surface. Kinetic analysis of site-directed mutants was carried out to determine the contributions made by Arg102, Arg150, Ser120, and Thr121 in binding the nucleotide unit. Lastly, we show by kinetic and X-ray analyses of Asp31, His64, and Glu78 site-directed mutants that these three active site residues are important for productive binding of the substrate 4-hydroxybenzoyl ring.     

Soluble cytochromes from the marine methanotroph Methylomonas sp. strain A4.

Soluble c-type cytochromes are central to metabolism of C1 compounds in methylotrophic bacteria. In order to characterize the role of c-type cytochromes in methane-utilizing bacteria (methanotrophs), we have purified four different cytochromes, cytochromes c-554, c-553, c-552, and c-551, from the marine methanotroph Methylomonas sp. strain A4. The two major species, cytochromes c-554 and c-552, were monoheme cytochromes and accounted for 57 and 26%, respectively, of the soluble c-heme. The approximate molecular masses were 8,500 daltons (Da) (cytochrome c-554) and 14,000 Da (cytochrome c-552), and the isoelectric points were pH 6.4 and 4.7, respectively. Two possible diheme c-type cytochromes were also isolated in lesser amounts from Methylomonas sp. strain A4, cytochromes c-551 and c-553. These were 16,500 and 34,000 Da, respectively, and had isoelectric points at pH 4.75 and 4.8, respectively. Cytochrome c-551 accounted for 9% of the soluble c-heme, and cytochrome c-553 accounted for 8%. All four cytochromes differed in their oxidized versus reduced absorption maxima and their extinction coefficients. In addition, cytochromes c-554, c-552, and c-551 were shown to have different electron paramagnetic spectra and N-terminal amino acid sequences. None of the cytochromes showed significant activity with purified methanol dehydrogenase in vitro, but our data suggested that cytochrome c-552 is probably the in vivo electron acceptor for the methanol dehydrogenase.     

Cyclic-imide-hydrolyzing activity of D-hydantoinase from Blastobacter sp. strain A17p-4.

The cyclic-imide-hydrolyzing activity of a prokaryotic cyclic-ureide-hydrolyzing enzyme, D-hydantoinase, was investigated. The enzyme hydrolyzed cyclic imides with bulky substituents such as 2-methylsuccinimide, 2-phenylsuccinimide, phthalimide, and 3,4-pyridine dicarboximide to the corresponding half-amides. However, simple cyclic imides without substituents, which are substrates of imidase (ie.g., succinimide, glutarimide, and sulfur-containing cyclic imides such as 2,4-thiazolidinedione and rhodanine), were not hydrolyzed. The combined catalytic actions of bacterial D-hydantoinase and imidase can cover the function of a single mammalian enzyme, dihydropyrimidinase. Prokaryotic D-hydantoinase also catalyzed the dehyrative cyclization of the half-amide phthalamidic acid to the corresponding cyclic imide, phthalimide. The reversible hydrolysis of cyclic imides shown by prokaryotic D-hydantoinase suggested that, in addition to pyrimidine metabolism, it may also function in cyclic-imide metabolism.     

Biodegradation of 4-nitrotoluene by Pseudomonas sp. strain 4NT.

A strain of Pseudomonas spp. was isolated from nitrobenzene-contaminated soil on 4-nitrotoluene as the sole source of carbon, nitrogen, and energy. The organism also grew on 4-nitrobenzaldehyde, and 4-nitrobenzoate. 4-Nitrobenzoate and ammonia were detected in the culture fluid of glucose-grown cells after induction with 4-nitrotoluene. Washed suspensions of 4-nitrotoluene- or 4-nitrobenzoate-grown cells oxidized 4-nitrotoluene, 4-nitrobenzaldehyde, 4-nitrobenzyl alcohol, and protocatechuate. Extracts from induced cells contained 4-nitrobenzaldehyde dehydrogenase, 4-nitrobenzyl alcohol dehydrogenase, and protocatechuate 4,5-dioxygenase activities. Under anaerobic conditions, cell extracts converted 4-nitrobenzoate or 4-hydroxylaminobenzoate to protocatechuate. Conversion of 4-nitrobenzoate to protocatechuate required NADPH. These results indicate that 4-nitrotoluene was degraded by an initial oxidation of the methyl group to form 4-nitrobenzyl alcohol, which was converted to 4-nitrobenzoate via 4-nitrobenzaldehyde. The 4-nitrobenzoate was reduced to 4-hydroxylaminobenzoate, which was converted to protocatechuate. A protocatechuate 4,5-dioxygenase catalyzed meta-ring fission of the protocatechuate. The detection of 4-nitrobenzaldehyde and 4-nitrobenzyl alcohol dehydrogenase and 4-nitrotoluene oxygenase activities in 4-nitrobenzoate-grown cells suggests that 4-nitrobenzoate is an inducer of the 4-nitrotoluene degradative pathway.     

Three-dimensional structure of the regularly constructed surface layer from Synechocystis sp. strain CLII

The isolated, outermost cell wall layer from Synechocystis sp. strain CLII is described using electron microscopy and Fourier reconstruction to study the three-dimensional structure of the proteins within the layer to a resolution of ca. 3 nm. This surface layer forms regular hexagonal arrays (a = b = 15.2 nm). The two-dimensional space group is p6. The monomer proteins form hexamers arranged around a central hollow cylinder. The linkers between the hexamers are of the delta type and are located approximately in the central section between the top and bottom of the protein layer.     

The solution structure of photosystem I accessory protein E from the cyanobacterium Nostoc sp. strain PCC 8009.

PsaE is a small basic subunit located on the stromal (cytoplasmic) side of photosystem I. In cyanobacteria, this subunit participates in cyclic electron transport and modulates the interactions of the complex with soluble ferredoxin. The PsaE protein isolated from the cyanobacterium Synechococcus sp. strain PCC 7002 adopts the beta topology of an SH3 domain, with five beta strands (betaA through betaE) and a turn of 3(10) helix between strands betaD and betaE [Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062]. The primary structure of the PsaE protein is strongly conserved across all oxygen-evolving photosynthetic organisms. However, variability in loop lengths, as well as N- or C-terminal extensions, suggests that the structure of a second representative PsaE subunit would be useful to characterize the interactions among photosystem I polypeptides. In this work, the solution structure of PsaE from the cyanobacterium Nostoc sp. strain PCC 8009 was determined by NMR methods. Compared to PsaE from Synechococcus sp. strain PCC 7002, this PsaE has a seven-residue deletion in the loop connecting strands betaC and betaD, and an eight-residue C-terminal extension. Angular and distance restraints derived from homonuclear and heteronuclear NMR experiments were used to calculate structures by a distance-geometry/simulated-annealing protocol. A family of 20 structures (rmsd of 0.24 A in the regular secondary structure) is presented. Differences between the two cyanobacterial proteins are mostly confined to the CD loop region; the C-terminal extension is disordered. The thermodynamic stability of Nostoc sp. strain PCC 8009 PsaE toward urea denaturation was measured by circular dichroism and fluorescence spectroscopy, and thermal denaturation was monitored by UV absorption spectroscopy. Chemical and thermal denaturation curves are modeled satisfactorily with two-state processes. The DeltaG degrees of unfolding at room temperature is 12.4 +/- 0.3 kJ mol(-1) (pH 5), and the thermal transition midpoint is 59 +/- 1 degrees C (pH 7). Interactions with other proteins in the photosystem I complex may aid in maintaining PsaE in its native state under physiological conditions.     

Inhibitors of the molybdenum cofactor containing 4-hydroxybenzoyl-CoA reductase

4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a member of the xanthine oxidase (XO) family of molybdenum cofactor containing enzymes and catalyzes the irreversible removal of a phenolic hydroxy group by reduction, yielding benzoyl-CoA and water. In this work the effects of various activity modulating compounds were characterized by kinetic, electron paramagnetic resonance (EPR) spectroscopic, and X-ray crystallographic studies. 4-HBCR was readily inactivated by cyanide and by the reducing agents titanium(III) citrate and dithionite; in contrast, reduced viologens had no inhibitory effect. Cyanide inhibition occurred in both the oxidized and reduced state of 4-HBCR. In the reduced state, cyanide-inhibited 4-HBCR was reactivated by simple oxidation. In contrast, reactivation from the oxidized state was only achieved in the presence of sulfide. Dithionite-inhibited 4-HBCR was reactivated by oxidation, whereas inhibition by titanium(III) citrate was irreversible. The previously reported inhibitory effect of azide could not be confirmed; instead, azide rather protected the enzyme from inactivation by titanium(III) citrate. The EPR spectra of the Mo(V) states were nearly identical in the noninhibited methyl viologen and in the dithionite-inhibited states of 4-HBCR; they exhibited a hyperfine splitting due to magnetic coupling with two solvent-exchangeable protons. The cyanide-treated enzyme showed the typical desulfo-inhibited Mo(V) EPR signal in D 2O, whereas in H 2O the hyperfine splitting was altered but indicated no loss of Mo(V)-proton interactions. The structures of dithionite- and azide-bound 4-HBCR were solved at 2.1 and 2.2 A, respectively. Both dithionite and azide bound directly to equatorial ligation sites of the Mo atom. The results obtained revealed further insights into the active site of an unusual member of the XO family of molybdenum cofactor containing enzymes.     

Primary structure of the peptidoglycan from the unicellular cyanobacterium Synechocystis sp. strain PCC 6714

A peptidoglycan fraction free of non-peptidoglycan components was isolated from the unicellular cyanobacterium Synechocystis sp. strain PCC 6714. Hydrofluoric acid treatment (48%, 0 degrees C, 48 h) cleaved off from the peptidoglycan non-peptidoglycan glucosamine, mannosamine, and mannose. The purified peptidoglycan consists of N-acetyl muramic acid, N-acetyl glucosamine, L-alanine, D-alanine, D-glutamic acid, and meso-diaminopimelic acid in approximately equimolar amounts. At least partial amidation of carboxy groups in the peptide subunits is indicated. Peptide analyses and 2,4-dinitrophenyl studies of partial acid hydrolysates revealed the structure of the Synechocystis sp. strain PCC 6714 peptidoglycan to belong to the A1 gamma type (direct cross-linkage) of peptidoglycan classification. The degree of cross-linkage is about 56% and thus is in the range of that found in gram-positive bacteria. Some of the peptide units are present as tripeptides lacking the carboxy-terminal D-alanine.     

X-ray crystal structure of benzoate 1,2-dioxygenase reductase from Acinetobacter sp. strain ADP1

One of the major processes for aerobic biodegradation of aromatic compounds is initiated by Rieske dioxygenases. Benzoate dioxygenase contains a reductase component, BenC, that is responsible for the two-electron transfer from NADH via FAD and an iron-sulfur cluster to the terminal oxygenase component. Here, we present the structure of BenC from Acinetobacter sp. strain ADP1 at 1.5 A resolution. BenC contains three domains, each binding a redox cofactor: iron-sulfur, FAD and NADH, respectively. The [2Fe-2S] domain is similar to that of plant ferredoxins, and the FAD and NADH domains are similar to members of the ferredoxin:NADPH reductase superfamily. In phthalate dioxygenase reductase, the only other Rieske dioxygenase reductase for which a crystal structure is available, the ferredoxin-like and flavin binding domains are sequentially reversed compared to BenC. The BenC structure shows significant differences in the location of the ferredoxin domain relative to the other domains, compared to phthalate dioxygenase reductase and other known systems containing these three domains. In BenC, the ferredoxin domain interacts with both the flavin and NAD(P)H domains. The iron-sulfur center and the flavin are about 9 A apart, which allows a fast electron transfer. The BenC structure is the first determined for a reductase from the class IB Rieske dioxygenases, whose reductases transfer electrons directly to their oxygenase components. Based on sequence similarities, a very similar structure was modeled for the class III naphthalene dioxygenase reductase, which transfers electrons to an intermediary ferredoxin, rather than the oxygenase component.     

Resolution of 4-chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS3 into three components.

Extracts of Pseudomonas sp. strain CBS3 grown with 4-chlorobenzoate as sole carbon source contained an enzyme that converted 4-chlorobenzoate to 4-hydroxybenzoate. This enzyme was shown to consist of three components, all necessary for the reaction. Component I, which had a molecular weight of about 3,000, was highly unstable. Components II and III were stable proteins with molecular weights of about 86,000 and 92,000.