Discovery and characterization of a putrescine oxidase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> NCIMB 11540

A gene encoding a putrescine oxidase (PuO_Rh, EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting of subunits of 50 kDa and contains non-covalently bound flavin adenine dinucleotide as a cofactor. From all substrates, the highest catalytic efficiency was found with putrescine (K _M = 8.2 μM, k _cat = 26 s⁻¹). PuO_Rh accepts longer polyamines, while short diamines and monoamines strongly inhibit activity. PuO_Rh is a reasonably thermostable enzyme with t _1/2 at 50°C of 2 h. Based on the crystal structure of human monoamine oxidase B, we constructed a model structure of PuO_Rh, which hinted to a crucial role of Glu324 for substrate binding. Mutation of this residue resulted in a drastic drop (five orders of magnitude) in catalytic efficiency. Interestingly, the mutant enzyme showed activity with monoamines, which are not accepted by wt-PuO_Rh. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An alternative flexible conformation of the <Emphasis Type="Italic">E. coli</Emphasis> HUβ<Subscript>2</Subscript> protein: structural,dynamics, and functional aspects

The histone-like HU protein is the major nucleoid-associated protein involved in the dynamics and structure of the bacterial chromosome. Under physiological conditions, the three possible dimeric forms of the E. coli HU protein (EcHUα₂, EcHUβ₂, and EcHUαβ) are in thermal equilibrium between two dimeric conformations (N₂ ↔ I₂) varying in their secondary structure content. High-temperature molecular dynamics simulations combined with NMR experiments provide information about structural and dynamics features at the atomic level for the N₂ to I₂ thermal transition of the EcHUβ₂ homodimer. On the basis of these data, a realistic 3D model is proposed for the major I₂ conformation of EcHUβ₂. This model is in agreement with previous experimental data.     

Yeast holoenzyme of protein kinase CK2 requires both β and β′ regulatory subunits for its activity

Protein kinase CK2 is a highly conserved Ser/Thr protein kinase that is ubiquitous among eucaryotic organisms and appears to play an important role in many cellular functions. This enzyme in yeast has a tetrameric structure composed of two catalytic (α and/or α′) subunits and two regulatory β and β′ subunits. Previously, we have reported isolation from yeast cells four active forms of CK2, composed of αα′ββ′, α₂ββ′, α′₂ββ′ and a free α′-catalytic subunit. Now, we report that in Saccharomyces cerevisiae CK2 holoenzyme regulatory β subunit cannot substitute other β′ subunit and only both of them can form fully active enzymatic unit. We have examined the subunit composition of tetrameric complexes of yeast CK2 by transformation of yeast strains containing single deletion of the β or β′ regulatory subunits with vectors carrying lacking CKB1 or CKB2 genes. CK2 holoenzyme activity was restored only in cases when both of them were present in the cell. Additional, co-immunoprecypitation experiments show that polyadenylation factor Fip1 interacts with catalytic α subunits of CK2 and interaction with beta subunits in the holoenzyme decreases CK2 activity towards this protein substrate. These data may help to elucidate the role of yeast protein kinase CK2β/β′ subunits in the regulation of holoenzyme assembly and phosphotransferase activity.     

Kinetic and structural studies on roles of the serine ligand and a strictly conserved tyrosine residue in nitrile hydratase

Nitrile hydratases (NHase), which catalyze the hydration of nitriles to amides, have an unusual Fe³⁺ or Co³⁺ center with two modified Cys ligands: cysteine sulfininate (Cys-SO₂ ⁻) and either cysteine sulfenic acid or cysteine sulfenate [Cys-SO(H)]. Two catalytic mechanisms have been proposed. One is that the sulfenyl oxygen activates a water molecule, enabling nucleophilic attack on the nitrile carbon. The other is that the Ser ligand ionizes the strictly conserved Tyr, activating a water molecule. Here, we characterized mutants of Fe-type NHase from Rhodococcus erythropolis N771, replacing the Ser and Tyr residues, αS113A and βY72F. The αS113A mutation partially affected catalytic activity and did not change the pH profiles of the kinetic parameters. UV–vis absorption spectra indicated that the electronic state of the Fe center was altered by the αS113A mutation, but the changes could be prevented by a competitive inhibitor, n-butyric acid. The overall structure of the αS113A mutant was similar to that of the wild type, but significant changes were observed around the catalytic cavity. Like the UV–vis spectra, the changes were compensated by the substrate or product. The Ser ligand is important for the structure around the catalytic cavity, but is not essential for catalysis. The βY72F mutant exhibited no activity. The structure of the βY72F mutant was highly conserved but was found to be the inactivated state, with αCys114-SO(H) oxidized to Cys-SO₂ ⁻, suggesting that βTyr72 affected the electronic state of the Fe center. The catalytic mechanism is discussed on the basis of the results obtained.     

A novel membrane-bound flavocytochrome c sulfide dehydrogenase from the colourless sulfur bacterium Thiobacillus sp. W5

A novel membrane-bound sulfide-oxidizing enzyme was purified 102-fold from the neutrophilic, obligately chemolithoautotrophic Thiobacillus sp. W5 by means of a six-step procedure. Spectral analysis revealed that the enzyme contains haem c and flavin. SDS-PAGE showed the presence of two types of subunit with molecular masses of 40 and 11 kDa. The smaller subunit contains covalently bound haem c, as was shown by haem staining. A combination of spectral analysis and the pyridine haemochrome test indicated that the sulfide-oxidizing heterodimer contains one molecule of haem c and one molecule of flavin. It appeared that the sulfide-oxidizing enzyme is a member of a small class of redox proteins, the flavocytochromes c, and is structurally most related to the flavocytochrome c sulfide dehydrogenase of the green sulfur bacterium Chlorobium limicola. The pH optimum of the enzyme is 8.6. At pH 9, the V _max was 2.1 ± 0.1 μmol cytochrome c (mg protein)^–1 min^–1, and the K _m values for sulfide and cytochrome c were 1.7 ± 0.4 μM and 3.8 ± 0.8 μM, respectively. Cyanide inhibited the enzyme by the formation of an N-5 adduct with the flavin moiety of the protein. On the basis of electron transfer stoichiometry, it seems likely that sulfur is the oxidation product. Received: 15 October 1996 / Accepted: 7 January 1997     

Structural and functional importance of theβ-turn in proteins. Studies on proline-containing peptides

We report here two sets of results on proline-containing linear peptides, one of which brings out the role of theβ-turn conformation in the structure of nascent collagen while the other points to the functional importance of the β-turn in calcium-binding proteins. Based on the data on peptides containing the -Pro-Gly-sequence, we had proposed and experimentally verified that theβ-turn conformation in these peptides is a structural requirement for the enzymic hydroxylation of the proline residues in the nascent (unhydroxylated) procollagen molecule. Our recent data, presented here, on the conformation of peptides containing both the -Pro-Gly- and -Gly-Pro-sequences reveal that while theβ-turn in the substrate molecule is required at the catalytic site of prolyl hydroxylase, the polyproline-II structure is necessary for effective binding at the active site of the enzyme. Thus, peptides containing either theβ-turn or the polyproline-II structure alone are found to act only as inhibitors while those with the polyproline-II followed byβ-turn serve as substrates of the enzyme. In another study, we have synthesized the two linear peptides: Boc-Pro-D-Ala-Ala-NHCH₃ and Boc-Pro-Gly-Ala-NHCH₃ each of which adopts, in solution, a structure with two consecutiveβ-turns, as judged from circular dichroism, infrared and nuclear magnetic resonance data. Drastic spectral changes are seen in these peptides on binding to Ca²⁺. Both the peptides show a distinct specificity to Ca²⁺ over Mg²⁺, Na⁺ and Li₊. A conformational change in the peptides occurs on Ca²⁺ binding which brings together the carbonyl groups to coordinate with the metal ion. These results imply a functional role for theβ-turn in Ca²⁺ — binding proteins.     

Influence of {\text{NH - }}{{\text{S}}^\gamma } bonding interactions on the structure and dynamics of metallothioneins

Mammalian metallothioneins ( \textM₇^\textIIMTs {\text{M}}_7^{\text{IIMTs}} ) show a clustered arrangement of the metal ions and a nonregular protein structure. The solution structures of Cd₃-thiolate cluster containing β-domain of mouse β-MT-1 and rat β-MT-2 show high structural similarities, but widely differing structure dynamics. Molecular dynamics simulations revealed a substantially increased number of \textNH - \textS^g {\text{NH - }}{{\text{S}}^\gamma } hydrogen bonds in β-MT-2, features likely responsible for the increased stability of the Cd₃-thiolate cluster and the enfolding protein domain. Alterations in the \textNH - \textS^g {\text{NH - }}{{\text{S}}^\gamma } hydrogen-bonding network may provide a rationale for the differences in dynamic properties encountered in the β-domains of MT-1, -2, and -3 isoforms, believed to be essential for their different biological function.     

Characteristics of protection by MgADP and MgATP of α3β3γ subcomplex of thermophilic Bacillus PS3 βY341W-mutant F1-ATPase from inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole support a bi-site mechanism of catalysis

MgADP and MgATP binding to catalytic sites of βY341W-α₃β₃Γ subcomplex of F₁-ATPase from thermophilic Bacillus PS3 has been assessed using their effect on the enzyme inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). It was assumed that NBD-Cl can inhibit only when catalytic sites are empty, and inhibition is prevented if a catalytic site is occupied with a nucleotide. In the absence of an activator, MgADP and MgATP protect βY341W-α₃β₃Γ sub-complex from inhibition by NBD-Cl by binding to two catalytic sites with an affinity of 37 μM and 12 mM, and 46 μM and 15 mM, respectively. In the presence of an activator lauryldimethylamine-N-oxide (LDAO), MgADP protects βY341W-α₃β₃Γ subcomplex from inhibition by NBD-Cl by binding to a catalytic site with a K _d of 12 mM. Nucleotide binding to a catalytic site with affinity in the millimolar range has not been previously revealed in the fluorescence quenching experiments with βY341W-α₃β₃Γ subcomplex. In the presence of activators LDAO or selenite, MgATP protects βY341W-α₃β₃Γ subcomplex from inhibition by NBD-Cl only partially, and the enzyme remains sensitive to inhibition by NBD-Cl even at MgATP concentrations that are saturating for ATPase activity. The results support a bi-site mechanism of catalysis by F₁-ATPases.     

Structure and mechanism of galactose oxidase: catalytic role of tyrosine 495

 The catalytic mechanism of the copper-containing enzyme galactose oxidase involves a protein radical on Tyr272, one of the equatorial copper ligands. The first step in this mechanism has been proposed to be the abstraction of a proton from the alcohol substrate by Tyr495, the axial copper ligand that is weakly co-ordinated to copper. In this study we have generated and studied the properties of a Y495F variant to test this proposal. X-ray crystallography reveals essentially no change from wild-type other than loss of the tyrosyl hydroxyl group. Visible spectroscopy indicates a significant change in the oxidised Y495F compared to wild-type with loss of a broad 810-nm peak, supporting the suggestion that this feature is due to inter-ligand charge transfer via the copper. The presence of a peak at 420 nm indicates that the Y495F variant remains capable of radical formation, a fact supported by EPR measurements. Thus the significantly reduced catalytic efficiency (1100-fold lower k _cat / K _m) observed for this variant is not due to an inability to generate the Tyr272 radical. By studying azide-induced pH changes, it is clear that the reduced catalytic efficiency is due mainly to the inability of Y495F to accept protons. This provides definitive evidence for the key role of Tyr495 in the initial proton abstraction step of the galactose oxidase catalytic mechanism. Received: 17 December 1996 / Accepted: 12 March 1997     

Cloning and Biochemical Characterization of BglC, a β-Glucosidase from the Cellulolytic Actinomycete Thermobifida fusca

An operon, bglABC, that encodes two sugar permeases and a β-glucosidase was cloned from a cellulolytic actinomycete, Thermobifida fusca, into Escherichia coli and sequenced. The bglC gene encoding an intracellular β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) belonging to glycosyl hydrolase family 1 was subcloned and expressed in E. coli. The purified enzyme (MW 53,407 Da; pI 4.69) hydrolyzed substrates containing both β 1 → 4 and β 1 → 2 glycosidic bonds, and was most active against cellobiose (V_max= 29, K _m = 0.34 mm), cellotriose, cellotetraose, and sophorose. The enzyme also showed aryl-β-glucosidase activity on p-nitrophenyl-β-d-glucopyranoside and p-nitrophenyl-β-d-cellobioside. BglC had a pH optimum of 7.0 and a temperature optimum of 50°C. The enzyme was stable at 60°C, but was rapidly inactivated at 65°C. BglC was inhibited by low concentrations of gluconolactone, but was insensitive to end-product inhibition by glucose and was not affected by Ca or Mg ions or EDTA. Its properties are well suited for use in a process to hydrolyze biomass cellulose to glucose. Received: 21 August 2000 / Accepted: 4 October 2000     

Comparative fluorescence studies of native and crosslinked glucose oxidase

The binding characteristics of flavin adenine dinucleotide (FAD) to apoenzyme preparations obtained from native and intramolecularly crosslinked glucose oxidase were determined and compared. The dissociation constants K_diss as well as rates of recombination of FAD with the two apoenzyme preparations, were independently evaluated from fluorescence quenching of either the tryptophans of FAD. The K_diss values thus obtained were <10^?19M for native glucose oxidase and 4 ± 1 × 10^?7M for the crosslinked enzyme. The recombination of apo glucose oxidase with FAD, which is presumably diffusion controlled, is followed by an apparent first order decrease in fluorescence intensity of both the protein tryptophans and FAD, with a rate constant around 0.2 min^?1. This could be related to conformational changes which occur immediately after binding of FAD to the apoenzyme, an interpretation which is supported by the markedly different results obtained in the analogous experiments with the crosslinked enzyme. A model for the conformational characteristics of glucose oxidase, based on this study, is proposed.     

Architecture and characterization of sarcosine oxidase from Thermococcus kodakarensis KOD1

Sarcosine oxidase (SOX) catalyzes the oxidation of the methyl group in sarcosine and transfer of the oxidized methyl group into the one-carbon metabolic pool. Here, we separately cloned and expressed α and β subunit of SOX from Thermococcus kodakarensis KOD1 (TkSOX) in Escherichia coli and the recombinant proteins were purified to homogeneity. Gel filtration chromatography and transmission electron microscopy analysis showed that the α subunit formed a dimeric structure and behaved as an NADH dehydrogenase; β subunit was a tetramer that had sarcosine oxidase and l-proline dehydrogenase activity. The TkSOX complex assembled into the hetero-octameric (αβ)₄ form and had NADH dehydrogenase activity. Gold-label analysis indicated that α and β subunits were oriented in the alternative form. Based on these results, we suggested that TkSOX was a multifunctional enzyme and that each subunit and (αβ)₄ complex may separately exist as a function enzyme in different conditions.     

Characterization of glycerol dehydratase expressed by fusing its α- and β-subunits

The gdh and gdhr genes, encoding B₁₂-dependent glycerol dehydratase (GDH) and glycerol dehydratase reactivase (GDHR), respectively, in Klebsiella pneumoniae, were cloned and expressed in E. coli. Part of the β-subunit was lost during GDH purification when co-expressing α, β and γ subunit. This was overcome by fusing the β-subunit to α- or γ-subunit with/without the insertion of a linker peptide between the fusion moieties. The kinetic properties of the fusion enzymes were characterized and compared with wild type enzyme. The results demonstrated that the fusion protein GDHALB/C, constructed by linking the N-terminal of β-subunit to the C-terminal of α subunit through a (Gly₄Ser)₄ linker peptide, had the greatest catalytic activity. Similar to the wild-type enzyme, GDHALB/C underwent mechanism-based inactivation by glycerol during catalysis and could be reactivated by GDHR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

X-ray absorption and diffraction studies of the metal binding sites in amyloid <Emphasis Type="Italic">β</Emphasis>-peptide

A major source of neurodegeneration observed in Alzheimer’s disease is believed to be caused by the toxicity from reactive oxygen species produced in the brain mediated by the Aβ protein and mainly copper species. An atomic model of an amyloid β-peptide (Aβ) Cu²⁺ complex or at least the structure of the metal binding site is of great interest. Accurate information about the Cu-binding site of Aβ protein can facilitate simulation of redox chemistry using high level quantum mechanics. Complementary X-ray diffraction and X-ray absorption techniques can be employed to obtain such accurate information. This review provides a blend of X-ray diffraction results on amyloid structures and selected works on Aβ Cu²⁺ binding based on spectroscopic measurements with emphasis on the X-ray absorption technique. Australian Society for Biophysics Special Issue: Metals and Membranes in Neuroscience.     

Glucose oxidase: natural occurrence, function, properties and industrial applications

Glucose oxidase (GOX) from Aspergillus niger is a well-characterised glycoprotein consisting of two identical 80-kDa subunits with two FAD co-enzymes bound. Both the DNA sequence and protein structure at 1.9 Ǻ have been determined and reported previously. GOX catalyses the oxidation of d-glucose (C₆H₁₂O₆) to d-gluconolactone (C₆H₁₀O₆) and hydrogen peroxide. GOX is produced naturally in some fungi and insects where its catalytic product, hydrogen peroxide, acts as an anti-bacterial and anti-fungal agent. GOX is Generally Regarded As Safe, and GOX from A. niger is the basis of many industrial applications. GOX-catalysed reaction removes oxygen and generates hydrogen peroxide, a trait utilised in food preservation. GOX has also been used in baking, dry egg powder production, wine production, gluconic acid production, etc. Its electrochemical activity makes it an important component in glucose sensors and potentially in fuel cell applications. This paper will give a brief background on the natural occurrence, functions as well as the properties of glucose oxidase. A good coverage on the diverse uses of glucose oxidase in the industry is presented with a brief outline on the working principles in the various settings. Furthermore, food grade GOX preparations are relatively affordable and widely available; the readers may be encouraged to explore other potential uses of GOX. One example is that GOX-catalysed reaction generates significant amount of heat (∼200 kJ/mol), and this property has been mostly neglected in the various applications described so far.     

Revisiting the Cellulosimicrobium cellulans yeast-lytic β-1,3-glucanases toolbox: A review

Cellulosimicrobium cellulans (also known with the synonyms Cellulomonas cellulans, Oerskovia xanthineolytica, and Arthrobacter luteus) is an actinomycete that excretes yeast cell wall lytic enzyme complexes containing endo-β-1,3-glucanases [EC 3.2.1.39 and 3.2.1.6] as key constituents. Three genes encoding endo-β-1,3-glucanases from two C. cellulans strains have been cloned and characterised over the past years. The βglII and βglII _A genes from strain DSM 10297 (also known as O. xanthineolytica LL G109) encoded proteins of 40.8 and 28.6 kDa, respectively, whereas the β-1,3-glucanase gene from strain ATCC 21606 (also known as A. luteus 73–14) encoded a 54.5 kDa protein. Alignment of their deduced amino acid sequences reveal that βglII and βglII _A have catalytic domains assigned to family 16 of glycosyl hydrolases, whereas the catalytic domain from the 54.5 kDa glucanase belongs to family 64. Notably, both βglII and the 54.5 kDa β-1,3-glucanase are multidomain proteins, having a lectin-like C-terminal domain that has been assigned to family 13 of carbohydrate binding modules, and that confers to β-1,3-glucanases the ability to lyse viable yeast cells. Furthermore, βglII may also undergo posttranslational proteolytic processing of its C-terminal domain, resulting in a truncated enzyme retaining its glucanase activity but with very low yeast-lytic activity. In this review, the diversity in terms of structural and functional characteristics of the C. cellulans β-1,3-glucanases has been compiled and compared.     

Structural and biochemical properties of glycosylated and deglycosylated glucose oxidase from Penicillium amagasakiense

Glucose oxidase from Penicillium amagasakiense was purified to homogeneity by ion-exchange chromatography and deglycosylated with endoglycosidase H. On the basis of gas chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoretic (SDS-PAGE) analyses, the protein-bound high-mannose-type carbohydrate moiety corresponded to 13% of the molecular mass of glycosylated glucose oxidase. A total of six N-glycosylation sites per dimer were determined from the N-acetylglucosamine content. The enzymatically deglycosylated enzyme contained less than 5% of the original carbohydrate moiety. A molecular mass of 130 kDa (gel filtration) and 133 kDa (native PAGE) was determined for the dimer and 67 kDa (SDS-PAGE) for the monomer of the deglycosylated enzyme. The N-terminal sequence, which has not been published for glucose oxidase from P. amagasakiense to date and which showed less than 50% homology to the N terminus of glucose oxidase from Aspergillus niger, and the amino acid composition were not altered by the deglycosylation. Deglycosylation also did not affect the kinetics of glucose oxidation or the pH and temperature optima. It also did not increase the susceptibility of the enzyme to proteolytic degradation. However, deglycosylated glucose oxidase exhibited decreased pH and thermal stability. The thermal stability of both enzymes was shown to be dependent on the buffer concentration and was enhanced by certain additives, particularly 1 M (NH₄)₂SO₄, which stabilised glucose oxidase 100- to 300-fold at 50 °C and pH 7–8, and 2 M KF, which stabilised the enzyme up to 36-fold at 60 °C and pH 6. In sodium acetate buffer, changes in pH (4–6) affected the affinity for glucose but had no effect on the V _max of the reaction. In contrast, in TRIS buffer, pH 8, a 10-fold decrease in V _max and a 2-fold decrease in K _m were observed. Received: 8 October 1996 / Received revision: 14 January 1997 / Accepted: 17 January 1997     

Purification and biochemical characterization of a transglucosilating β-glucosidase of <Emphasis Type="Italic">Stachybotrys</Emphasis> strain

The filamentous fungus Stachybotrys sp has been shown to possess a rich β-glucosidase system composed of five β-glucosidases. One of them was already purified to homogeneity and characterized. In this work, a second β-glucosidase was purified and characterized. The filamentous fungal A19 strain was fed-batch cultivated on cellulose, and its extracellular cellulases (mainly β-glucosidases) were analyzed. The purified enzyme is a monomeric protein of 78 kDa molecular weight and exhibits optimal activity at pH 6.0 and at 50°C. The kinetic parameters, K _m and V _max, on para-nitro-phenyl-β-d-glucopyranosid (p-NPG) as a substrate were, respectively, 1.846 ± 0.11 mM and 211 ± 0.08 μmol min⁻¹ ml⁻¹. One interesting feature of this enzyme is its high stability in a wide range of pH from 4 to 10. Besides its aryl β-glucosidase activity towards salicin, methylumbellypheryl-β-d-glucoside (MU-Glc), and p-NPG, it showed a true β-glucosidase activity because it splits cellobiose into two glucose monomers. This enzyme has the capacity to synthesize short oligosaccharides from cellobiose as the substrate concentration reaches 30% with a recovery of 40%. We give evidences for the involvement of a transglucosylation to synthesize cellotetraose by a sequential addition of glucose to cellotriose.     

Expression and homology modeling of 2′-aminobiphenyl-2,3-diol-1,2-dioxygenase from Pseudomonas stutzeri carbazole degradation pathway

The enzyme 2′-aminobiphenyl-2,3-diol-1,2-dioxygenase (CarB), encoded by two genes (carBa and carBb), is an α₂β₂ heterotetramer that presents meta-cleavage activity toward the hydroxylated aromatic ring in the carbazole degradation pathway from petroleum-degrader bacteria Pseudomonas spp. The 1082-base, pair polymerase chain reaction product corresponding to, carBaBb genes from Pseudomonas stutzeri ATCC 31258 was cloned by site-specific recombination and expressed in high levels in Escherichia coli BL21-SI with a histidine-tag and in native form. The CarB activity toward 2,3-dihydroxybiphenyl was similar for these two constructions. The α₂β₂ 3D model of CarB dioxygenase was proposed by homology modeling using the protocatechuate 4,5-dioxygenase (LigAB) structure as template. Accordingly, His12, His53, and Glu230 coordinate the Fe(II) in the catalytic site at the subunit CarBb. The model also indicates that His182 is the catalytic base responsible for deprotonating one of the hydroxyl group of the substrate by a hydrogen bond. The hydrophobic residues Trp257 and Phe258 in the CarB structure substituted the LigAB amino acid residues Ser269 and Asn270. These data could explain why the CarB was active for 2,3-dihydroxybiphenyl and not for protocatechuate.