Lowering pH optimum of activity of SshEstI, a slightly alkaliphilic archaeal esterase of the hormone-sensitive lipase family.

SshEstI, a carboxylesterase from the thermoacidophilic archaeon Saccharolobus shibatae, is a member of the hormone-sensitive lipase family that displays slightly alkaliphilic activity with an optimum activity at pH 8.0. In this study, three distinct strategies were explored to confer acidophilic properties to SshEstI. The first strategy involved engineering the oxyanion hole by replacing Gly81 with serine or aspartic acid. The G81S mutant showed optimum activity at pH 7.0, whereas the aspartic acid mutant (G81D) rendered the enzyme slightly acidophilic with optimum activity observed at pH 6.0; however, kcat and kcat/Km values were reduced by these substitutions. The second strategy involved examining the effects of surfactant additives on the pH-activity profiles of SshEstI. The results showed that cetyltrimethylammonium bromide (CTAB) enhanced wild-type enzyme (WT) activity at acidic pH values. In the presence of 0.1 mM CTAB, G81S and G81D were acidophilic enzymes with optimum activity at pH 6.0 and 4.0, respectively, although their enzyme activities were low. The third strategy involved engineering the active site to resemble that of kumamolisin-As (kuma-As), an acidophilic peptidase of the sedolisin family. The catalytic triad of kuma-As was exchanged into SshEstI using site-directed mutagenesis. X-ray crystallographic analysis of the mutants (H274D and H274E) revealed that the potential hydrogen donor-acceptor distances around the active site of WT were fully maintained in these mutants. However, these mutants were inactive at pH 4-8.

A retrospect of the structure determination of Taka-amylase A.

The 3D structure of Taka-amylase A was determined by X-ray crystal analysis at 3 Å resolution by Masao Kakudo's laboratory at the Institute for Protein Research, Osaka University, in 1980. Seven kinds of heavy atom derivatives were used for phase determination. There are three copies of Taka-amylase molecules in the asymmetric unit, which improved the quality of electron density maps, leading to the completion of a molecular model with 478 amino acids. The structure determination process in those days is described briefly.

Structural analysis and reaction mechanism of the disproportionating enzyme (D-enzyme) from potato.

Starch produced by plants is a stored form of energy and is an important dietary source of calories for humans and domestic animals. Disproportionating enzyme (D-enzyme) catalyzes intramolecular and intermolecular transglycosylation reactions of α-1, 4-glucan. D-enzyme is essential in starch metabolism in the potato. We present the crystal structures of potato D-enzyme, including two different types of complex structures: a primary Michaelis complex (substrate binding mode) for 26-meric cycloamylose (CA26) and a covalent intermediate for acarbose. Our study revealed that the acarbose and CA26 reactions catalyzed by potato D-enzyme involve the formation of a covalent intermediate with the donor substrate. HPAEC of reaction substrates and products revealed the activity of the potato D-enzyme on acarbose and CA26 as donor substrates. The structural and chromatography analyses provide insight into the mechanism of the coupling reaction of CA and glucose catalyzed by the potato D-enzyme. The enzymatic reaction mechanism does not involve residual hydrolysis. This could be particularly useful in preventing unnecessary starch degradation leading to reduced crop productivity. Optimization of this mechanism would be important for improvements of starch storage and productivity in crops.

Crystal structure of the starch-binding domain of glucoamylase from Aspergillus niger.

Glucoamylases are widely used commercially to produce glucose syrup from starch. The starch-binding domain (SBD) of glucoamylase from Aspergillus niger is a small globular protein containing a disulfide bond. The structure of A. niger SBD has been determined by NMR, but the conformation surrounding the disulfide bond was unclear. Therefore, X-ray crystal structural analysis was used to attempt to clarify the conformation of this region. The SBD was purified from an Escherichia coli-based expression system and crystallized at 293 K. The initial phase was determined by the molecular-replacement method, and the asymmetric unit of the crystal contained four protomers, two of which were related by a noncrystallographic twofold axis. Finally, the structure was solved at 2.0 Šresolution. The SBD consisted of seven ß-strands and eight loops, and the conformation surrounding the disulfide bond was determined from a clear electron-density map. Comparison of X-ray- and NMR-determined structures of the free SBD showed no significant difference in the conformation of each ß-strand, but the conformations of the loops containing the disulfide bond and the L5 loop were different. In particular, the difference in the position of the Cα atom of Cys509 between the X-ray- and NMR-determined structures was 13.3 Å. In addition, the B factors of the amino-acid residues surrounding the disulfide bond are higher than those of other residues. Therefore, the conformation surrounding the disulfide bond is suggested to be highly flexible.

Atomic structure of an archaeal GAN suggests its dual roles as an exonuclease in DNA repair and a CMG component in DNA replication.

In eukaryotic DNA replication initiation, hexameric MCM (mini-chromosome maintenance) unwinds the template double-stranded DNA to form the replication fork. MCM is activated by two proteins, Cdc45 and GINS, which constitute the 'CMG' unwindosome complex together with the MCM core. The archaeal DNA replication system is quite similar to that of eukaryotes, but only limited knowledge about the DNA unwinding mechanism is available, from a structural point of view. Here, we describe the crystal structure of an archaeal GAN (GINS-associated nuclease) from Thermococcus kodakaraensis, the homolog of eukaryotic Cdc45, in both the free form and the complex with the C-terminal domain of the cognate Gins51 subunit (Gins51C). This first archaeal GAN structure exhibits a unique, 'hybrid' structure between the bacterial RecJ and the eukaryotic Cdc45. GAN possesses the conserved DHH and DHH1 domains responsible for the exonuclease activity, and an inserted CID (CMG interacting domain)-like domain structurally comparable to that in Cdc45, suggesting its dual roles as an exonuclease in DNA repair and a CMG component in DNA replication. A structural comparison of the GAN-Gins51C complex with the GINS tetramer suggests that GINS uses the mobile Gins51C as a hook to bind GAN for CMG formation.

Involvement of FKBP6 in hepatitis C virus replication.

The chaperone system is known to be exploited by viruses for their replication. In the present study, we identified the cochaperone FKBP6 as a host factor required for hepatitis C virus (HCV) replication. FKBP6 is a peptidyl prolyl cis-trans isomerase with three domains of the tetratricopeptide repeat (TPR), but lacks FK-506 binding ability. FKBP6 interacted with HCV nonstructural protein 5A (NS5A) and also formed a complex with FKBP6 itself or FKBP8, which is known to be critical for HCV replication. The Val(121) of NS5A and TPR domains of FKBP6 were responsible for the interaction between NS5A and FKBP6. FKBP6 was colocalized with NS5A, FKBP8, and double-stranded RNA in HCV-infected cells. HCV replication was completely suppressed in FKBP6-knockout hepatoma cell lines, while the expression of FKBP6 restored HCV replication in FKBP6-knockout cells. A treatment with the FKBP8 inhibitor N-(N', N'-dimethylcarboxamidomethyl)cycloheximide impaired the formation of a homo- or hetero-complex consisting of FKBP6 and/or FKBP8, and suppressed HCV replication. HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue. These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A.

Structural insights into the low pH adaptation of a unique carboxylesterase from Ferroplasma: altering the pH optima of two carboxylesterases.

To investigate the mechanism for low pH adaptation by a carboxylesterase, structural and biochemical analyses of EstFa_R (a recombinant, slightly acidophilic carboxylesterase from Ferroplasma acidiphilum) and SshEstI (an alkaliphilic carboxylesterase from Sulfolobus shibatae DSM5389) were performed. Although a previous proteomics study by another group showed that the enzyme purified from F. acidiphilum contained an iron atom, EstFa_R did not bind to iron as analyzed by inductively coupled plasma MS and isothermal titration calorimetry. The crystal structures of EstFa_R and SshEstI were determined at 1.6- and 1.5-Å resolutions, respectively. EstFa_R had a catalytic triad with an extended hydrogen bond network that was not observed in SshEstI. Quadruple mutants of both proteins were created to remove or introduce the extended hydrogen bond network. The mutation on EstFa_R enhanced its catalytic efficiency and gave it an alkaline pH optimum, whereas the mutation on SshEstI resulted in opposite effects (i.e. a decrease in the catalytic efficiency and a downward shift in the optimum pH). Our experimental results suggest that the low pH optimum of EstFa_R activity was a result of the unique extended hydrogen bond network in the catalytic triad and the highly negatively charged surface around the active site. The change in the pH optimum of EstFa_R happened simultaneously with a change in the catalytic efficiency, suggesting that the local flexibility of the active site in EstFa_R could be modified by quadruple mutation. These observations may provide a novel strategy to elucidate the low pH adaptation of serine hydrolases.

Crystallization and preliminary X-ray diffraction analysis of domain-chimeric L-(2S,3S)-butanediol dehydrogenase.

A domain-chimeric L-2,3-butanediol dehydrogenase (chimera L-BDH), which was designed to possess both the S-configuration specificity of L-BDH and the stability of meso-BDH, was constructed by exchanging the respective domains of these two BDHs. However, chimera L-BDH possessed a lower enzymatic function than expected based on the two original enzymes. To elucidate the causes of the decreased stability and substrate specificity, crystallization of the protein was performed. Chimera L-BDH was purified to homogeneity via ammonium sulfate fractionation and three column-chromatography steps, and was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group C2221, diffracted synchrotron radiation to 1.58 Šresolution and were most likely to contain two molecules in the asymmetric unit.

Modification of chimeric (2S, 3S)-butanediol dehydrogenase based on structural information.

A chimeric (2S, 3S)-butanediol dehydrogenase (cLBDH) was engineered to have the strict (S)-configuration specificity of the (2S, 3S)-BDH (BsLBDH) derived from Brevibacterium saccharolyticum as well as the enzymatic stability of the (2R, 3S)-BDH (KpMBDH) from Klebsiella pneumonia by swapping the domains of two native BDHs. However, while cLBDH possesses the stability, it lacks the specificity. In order to assist in the design a BDH having strict substrate specificity, an X-ray structural analysis of a cLBDH crystal was conducted at 1.58 Å. The results obtained show some readily apparent differences around the active sites of cLBDH and BsLBDH. Based on this structural information, a novel (2S, 3S)-BDH having a preferred specificity was developed by introducing a V254L mutation into cLBDH. The influence of this mutation on the stability of cLBDH was not evaluated. Nevertheless, the technique described herein is an effective method for the production of a tailor-made BDH.

Crystal structure analysis of a recombinant predicted acetamidase/formamidase from the thermophile Thermoanaerobacter tengcongensis.

The structure of acetamidase/formamidase (Amds/Fmds) from the archaeon Thermoanaerobacter tengcongensis has been determined by X-ray diffraction analysis using MAD data in a crystal of space group P21, with unit-cell parameters a = 41.23 (3), b = 152.88 (6), c = 100.26 (7) Å, ß = 99.49 (3) ° and been refined to a crystallographic R-factor of 17.4% and R-free of 23.7%. It contains two dimers in one asymmetric unit, in which native Amds/Fmds (TE19) contains of the 32 kDa native protein. The final model consists of 4 monomer (299 amino acids residues with additional 2 expression tag amino acids residues), 5 Ca²âº, 4 Zn²âº and 853 water molecules. The monomer is composed by the following: an N-domain which is featuring by three-layers ß/ß/ß; a prominent excursion between N-terminal end of strand ß7 and ß11, which contains four-stranded antiparallel ß sheet; an C-domain which is formed by the last 82 amino acid residues with the feature of mixed α/ß structure. The protein contains ion-pair Ca²âº-Zn²âº. The portion of three-layer ß/ß/ß along with the loops provides four protein ligands to the tightly bound Ca²âº, three water molecules complete the coordination; and provides five protein ligands to the tightly bound Zn²âº, one water molecule complete the coordination.

Variation and predicted structure of the flagellin gene in Actinoplanes species.

Members of the genus Actinoplanes are considered to be representative of motile actinomycetes. To infer the flagellar diversity of Actinoplanes species, novel degenerate primers were designed for the flagellin (fliC) gene. The fliC gene of 21 Actinoplanes strains was successfully amplified and classified into two groups based on whether they were large (type I) or small (type II). Comparison of the translated amino acid sequences revealed that this size difference could be attributed to large number of gaps located in the central variable region. However, the C- and N- terminal regions were conserved. Except for a region on the flagellum surface, structural predictions of type I and II flagellins revealed that the two flagellin types were strongly correlated with each other. Phylogenetic analysis of the 115-amino acid N-terminal sequences revealed that the Actinoplanes species formed three clusters, and type II flagellin gene containing three type strains were phylogenetically closely related each other.

Structure-based design, synthesis, and evaluation of peptide-mimetic SARS 3CL protease inhibitors.

The design and evaluation of low molecular weight peptide-based severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CL) protease inhibitors are described. A substrate-based peptide aldehyde was selected as a starting compound, and optimum side-chain structures were determined, based on a comparison of inhibitory activities with Michael type inhibitors. For the efficient screening of peptide aldehydes containing a specific C-terminal residue, a new approach employing thioacetal to aldehyde conversion mediated by N-bromosuccinimide was devised. Structural optimization was carried out based on X-ray crystallographic analyses of the R188I SARS 3CL protease in a complex with each inhibitor to provide a tetrapeptide aldehyde with an IC(50) value of 98 nM. The resulting compound carried no substrate sequence, except for a P(3) site directed toward the outside of the protease. X-ray crystallography provided insights into the protein-ligand interactions.

Steric hindrance by 2 amino acid residues determines the substrate specificity of isomaltase from Saccharomyces cerevisiae.

The structures of the E277A isomaltase mutant from Saccharomyces cerevisiae in complex with isomaltose or maltose were determined at resolutions of 1.80 and 1.40Å, respectively. The root mean square deviations between the corresponding main-chain atoms of free isomaltase and the E277Α-isomaltose complex structures and those of free isomaltase and the E277A-maltose complex structures were found to be 0.131Å and 0.083Å, respectively. Thus, the amino acid substitution and ligand binding do not affect the overall structure of isomaltase. In the E277A-isomaltose structure, the bound isomaltose was readily identified by electron densities in the active site pocket; however, the reducing end of maltose was not observed in the E277A-maltose structure. The superposition of maltose onto the E277A-maltose structure revealed that the reducing end of maltose cannot bind to the subsite +1 due to the steric hindrance from Val216 and Gln279. The amino acid sequence comparisons with α-glucosidases showed that a bulky hydrophobic amino acid residue is conserved at the position of Val216 in α-1,6-glucosidic linkage hydrolyzing enzymes. Similarly, a bulky amino acid residue is conserved at the position of Gln279 in α-1,6-glucosidic linkage-only hydrolyzing α-glucosidases. Ala, Gly, or Asn residues were located at the position of α-1,4-glucosidic linkage hydrolyzing α-glucosidases. Two isomaltase mutant enzymes - V216T and Q279A - hydrolyzed maltose. Thus, the amino acid residues at these positions may be largely responsible for determining the substrate specificity of α-glucosidases.

S1-state Mn4Ca complex of Photosystem II exists in equilibrium between the two most-stable isomeric substates: XRD and EXAFS evidence.

Photosynthetic water oxidation reaction driven by Sun and catalyzed by a unique Mn(4)Ca cluster in Photosystem II (PSII) is known to take place in an oxygen evolving complex (OEC) that cycles five serial redox states, named "Kok's S(i)-states" (i=0-4). Recently, the atomic crystal structure of PSII from Thermosynechococcus vulcanus was resolved by 1.9 Å-resolution XRD data [55]. Interestingly, it revealed an unusual oxo-bridged Mn(4)CaO(5) cluster in the dark stable S(1)-state, e.g. unusual mono-µ(2)-oxo-mono-µ(4)-oxo-mono-µ(2)-carboxylato bridges connecting Mn(a) (terminal) and Mn(b) (central) ions with unusual atomic distance of 2.9 Å. Using the UDFT/B3LYP/lacvp** geometry optimization method and a truncated cluster model of the chemically-complete OEC put in ε=4 dielectric medium, it is shown that the OEC in S(1) must be in thermal equilibrium between the most-stable isomeric substates ("S(1a) and S(1b)") owing to the quasi-reversible structure change induced by proton migration. Coincidentally, it is found that the Mn(a)-Mn(b) distances in the Mn(4)Ca clusters in S(1a) and S(1b) are given by R(ab)=3.32 Å and 2.77 Å, respectively, so that the apparent distance between Mn(a) and Mn(b) ions in isomeric equilibrium is given by 2.94 Å, in agreement with experimental R(ab)~2.9 Å. Concomitantly, the first full-k-range EXAFS spectrum from powdered PSII [45] is used to provide the second experimental evidence for the S(1)-state OEC being in thermal equilibrium between S(1a) and S(1b)-isomers. These OEC-isomers consist of all the chemically-essential 11 amino acid residues, six cofactor ions and nine essential hydrated water molecules in their chemical ionic states around physiological pH 7, thus reasonably satisfying the biochemical charge neutrality with four Mn ions staying at the oxidation states (Mn(a)(III)/Mn(b)(IV)/Mn(c)(III)/Mn(d)(IV)) with the skeleton structures of MT-5J type and T-shaped DD-4J type. These H-bonding water molecules are found to fill a cavity connecting possible substrate/products channels so as to be arranged as an indispensable part of the catalytic Mn(4)Ca cluster in the order of "current-substrates" (W1/W2 bound to Mn(a)(III)), "next-substrates" (W4/W7) and "next-after-next-substrates" (W5/W6 bound to Ca(2+)). Results show that the Jahn-Teller effect due to Mn(a)(III) ion in these isomers can reasonably explain the very-slow-exchange and very-fast-exchange processes observed in S(1) by time-resolved (18)O-exchange mass spectroscopy.

Construction of a tailor-made L (2S,3S)-butanediol dehydrogenase by exchanging domains between native structural analogs.

The development of a stable L-BDH chimera was attempted by exchanging whole domains between two native structural analogs, L-BDH and meso-BDH, because the S-configuration specificity of L-BDH is valuable from the standpoint of its application but its activity is unstable, whereas meso-BDH is stable. The domain chimeras obtained indicated that the leaf-like structures constituting three domains were likely to be mainly associated with chiral recognition, and the fourth domain, the basic domain, is likely to be mainly associated with enzyme stability. A combination of the leaf domains of L-BDH and the basic domain of meso-BDH attained a sufficient level of practical use as an artificial L-BDH chimera, because the resulting enzyme had both stability and S-configuration specificity. However, the levels of stability and specificity were slightly lower than those of the respective enzymes from which they were derived.

Galactose recognition by a tetrameric C-type lectin, CEL-IV, containing the EPN carbohydrate recognition motif.

CEL-IV is a C-type lectin isolated from a sea cucumber, Cucumaria echinata. This lectin is composed of four identical C-type carbohydrate-recognition domains (CRDs). X-ray crystallographic analysis of CEL-IV revealed that its tetrameric structure was stabilized by multiple interchain disulfide bonds among the subunits. Although CEL-IV has the EPN motif in its carbohydrate-binding sites, which is known to be characteristic of mannose binding C-type CRDs, it showed preferential binding of galactose and N-acetylgalactosamine. Structural analyses of CEL-IV-melibiose and CEL-IV-raffinose complexes revealed that their galactose residues were recognized in an inverted orientation compared with mannose binding C-type CRDs containing the EPN motif, by the aid of a stacking interaction with the side chain of Trp-79. Changes in the environment of Trp-79 induced by binding to galactose were detected by changes in the intrinsic fluorescence and UV absorption spectra of WT CEL-IV and its site-directed mutants. The binding specificity of CEL-IV toward complex oligosaccharides was analyzed by frontal affinity chromatography using various pyridylamino sugars, and the results indicate preferential binding to oligosaccharides containing Galß1-3/4(Fucα1-3/4)GlcNAc structures. These findings suggest that the specificity for oligosaccharides may be largely affected by interactions with amino acid residues in the binding site other than those determining the monosaccharide specificity.

Crystal structures of isomaltase from Saccharomyces cerevisiae and in complex with its competitive inhibitor maltose.

The structures of isomaltase from Saccharomyces cerevisiae and in complex with maltose were determined at resolutions of 1.30 and 1.60 Å, respectively. Isomaltase contains three domains, namely, A, B, and C. Domain A consists of the (ß/α)(8) -barrel common to glycoside hydrolase family 13. However, the folding of domain C is rarely seen in other glycoside hydrolase family 13 enzymes. An electron density corresponding to a nonreducing end glucose residue was observed in the active site of isomaltase in complex with maltose; however, only incomplete density was observed for the reducing end. The active site pocket contains two water chains. One water chain is a water path from the bottom of the pocket to the surface of the protein, and may act as a water drain during substrate binding. The other water chain, which consists of six water molecules, is located near the catalytic residues Glu277 and Asp352. These water molecules may act as a reservoir that provides water for subsequent hydrolytic events. The best substrate for oligo-1,6-glucosidase is isomaltotriose; other, longer-chain, oligosaccharides are also good substrates. However, isomaltase shows the highest activity towards isomaltose and very little activity towards longer oligosaccharides. This is because the entrance to the active site pocket of isomaltose is severely narrowed by Tyr158, His280, and loop 310-315, and because the isomaltase pocket is shallower than that of other oligo-1,6-glucosidases. These features of the isomaltase active site pocket prevent isomalto-oligosaccharides from binding to the active site effectively.

Production of tetraketide lactones by mutated Antirrhinum majus chalcone synthases (AmCHS1).

Chalcone synthase (CHS) is a key enzyme of flavonoid biosynthesis in higher plants, catalyzing the stepwise decarboxylative condensation of three acetate units from malonyl-CoA with p-coumaroyl-CoA to yield 2',4,4',6'-tetrahydroxychalcone (THC). Reaction (at pH 7.5) of a mutant (V196M/T197A) of Antirrhinum majus CHS (AmCHS1) with p-coumaroyl-CoA and malonyl-CoA yielded a significant amount of a non-chalcone product, along with a small amount of THC. The non-chalcone product was identified as p-coumaroyltriacetic acid lactone (CTAL), a tetraketide lactone produced due to derailment from the canonical THC-producing reaction pathway. In vitro, the wild-type AmCHS1 showed low CTAL-producing activity at pH 7.5, but an appreciable level at pH 10. Each of the amino acid substitutions, V196M, T197A and V196M/T197A, caused a shift toward neutrality of the optimum pH for CTAL-producing activity. The V196M substitution resulted in a loss of THC-producing activity, as well as a 12.6-fold enhancement of CTAL-producing activity (at pH 7.5); hence, AmCHS1 was converted to a p-coumaroyltriacetic acid synthase by this single amino acid substitution. The THC-producing activity of the V196M mutant appeared to be restored by additional T197A substitution, although a single T197A substitution caused no substantial enhancement of the CTAL-producing activity of the wild-type enzyme. The enhancement of the tetraketide producing activity upon V196M and V196M/T197A substitutions was most markedly observed when p-coumaroyl-CoA was used as the starter substrate, and only slightly with benzoyl-, caffeoyl- and hexanoyl-CoAs. These results show the importance of the two contiguous amino acids at positions 196 and 197 for product specificity of an AmCHS1-catalyzed reaction.

Structural basis for chiral substrate recognition by two 2,3-butanediol dehydrogenases.

2,3-butanediol dehydrogenase (BDH) catalyzes the NAD-dependent redox reaction between acetoin and 2,3-butanediol. There are three types of homologous BDH, each stereospecific for both substrate and product. To establish how these homologous enzymes possess differential stereospecificities, we determined the crystal structure of l-BDH with a bound inhibitor at 2.0 A. Comparison with the inhibitor binding mode of meso-BDH highlights the role of a hydrogen-bond from a conserved Trp residue(192). Site-directed mutagenesis of three active site residues of meso-BDH, including Trp(190), which corresponds to Trp(192) of L-BDH, converted its stereospecificity to that of L-BDH. This result confirms the importance of conserved residues in modifying the stereospecificity of homologous enzymes.

New role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.

Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.