Arsenate reduction: thiol cascade chemistry with convergent evolution

The frequent abundance of arsenic in the environment has guided the evolution of enzymes for the reduction of arsenate. The arsenate reductases (ArsC) from different sources have unrelated sequences and structural folds, and can be divided into different classes on the basis of their structures, reduction mechanisms and the locations of catalytic cysteine residues. The thioredoxin-coupled arsenate reductase class is represented by Staphylococcus aureus pI258 ArsC and Bacillus subtilis ArsC. The ArsC from Escherichia coli plasmid R773 and the eukaryotic ACR2p reductase from Saccharomyces cerevisiae represent two distinct glutaredoxin-linked ArsC classes. All are small cytoplasmic redox enzymes that reduce arsenate to arsenite by the sequential involvement of three different thiolate nucleophiles that function as a redox cascade. In contrast, the ArrAB complex is a bacterial heterodimeric periplasmic or a surface-anchored arsenate reductase that functions as a terminal electron acceptor and transfers electrons from the membrane respiratory chain to arsenate. Finally, the less well documented arsenate reductase activity of the monomeric arsenic(III) methylase, which is an S-adenosylmethionine (AdoMet)-dependent methyltransferase. After each oxidative methylation cycle and before the next methylation step, As(V) is reduced to As(III). Methylation by this enzyme is also considered an arsenic-resistance mechanism for bacteria, fungi and mammals.     

Arginine 60 in the ArsC arsenate reductase of E. coli plasmid R773 determines the chemical nature of the bound As(III) product

Arsenic is a ubiquitous environmental toxic metal. Consequently, organisms detoxify arsenate by reduction to arsenite, which is then excreted or sequestered. The ArsC arsenate reductase from Escherichia coli plasmid R773, the best characterized arsenic-modifying enzyme, has a catalytic cysteine, Cys 12, in the active site, surrounded by an arginine triad composed of Arg 60, Arg 94, and Arg 107. During the reaction cycle, the native enzyme forms a unique monohydroxyl Cys 12-thiol-arsenite adduct that contains a positive charge on the arsenic. We hypothesized previously that this unstable intermediate allows for rapid dissociation of the product arsenite. In this study, the role of Arg 60 in product formation was evaluated by mutagenesis. A total of eight new structures of ArsC were determined at resolutions between 1.3 A and 1.8 A, with R(free) values between 0.18 and 0.25. The crystal structures of R60K and R60A ArsC equilibrated with the product arsenite revealed a covalently bound Cys 12-thiol-dihydroxyarsenite without a charge on the arsenic atom. We propose that this intermediate is more stable than the monohydroxyarsenite intermediate of the native enzyme, resulting in slow release of product and, consequently, loss of activity.     

The conserved glycine/alanine residue of the active-site loop containing the putative acetylCoA-binding motif is essential for the overall structural integrity of Mesorhizobium loti arylamine N-acetyltransferase 1

The arylamine N-acetyltransferases are important xenobiotic-metabolizing enzymes that catalyze an acetyl group transfer from acetylCoA to arylamine substrates. NAT enzymes possess an active-site loop (the active-site P-loop) involved in substrate binding and selectivity. The Gly/Ala residue present at the start of the active-site P-loop, although conserved in all NAT enzymes, is not involved in the catalytic mechanism or substrate binding. Here we show that a small amino acid (such as Gly or Ala) at this position is important not only for maintaining the functions of the active-site P-loop but, more surprisingly, also important for maintaining the overall structural integrity of NAT enzymes. Our data thus suggest that in addition to its role in substrate binding and selectivity, the active-site P-loop could play a wider structural role in NAT enzymes.     

How thioredoxin can reduce a buried disulphide bond

We present a study of the interaction between thioredoxin and the model enzyme pI258 arsenate reductase (ArsC) from Staphylococcus aureus. ArsC catalyses the reduction of arsenate to arsenite. Three redox active cysteine residues (Cys10, Cys82 and Cys89) are involved. After a single catalytic arsenate reduction event, oxidized ArsC exposes a disulphide bridge between Cys82 and Cys89 on a looped-out redox helix. Thioredoxin converts oxidized ArsC back towards its initial reduced state. In the absence of a reducing environment, the active-site P-loop of ArsC is blocked by the formation of a second disulphide bridge (Cys10-Cys15). While fully reduced ArsC can be recovered by exposing this double oxidized ArsC to thioredoxin, the P-loop disulphide bridge is itself inaccessible to thioredoxin. To reduce this buried Cys10-Cys15 disulphide-bridge in double oxidized ArsC, an intra-molecular Cys10-Cys82 disulphide switch connects the thioredoxin mediated inter-protein thiol-disulphide transfer to the buried disulphide. In the initial step of the reduction mechanism, thioredoxin appears to be selective for oxidized ArsC that requires the redox helix to be looped out for its interaction. The formation of a buried disulphide bridge in the active-site might function as protection against irreversible oxidation of the nucleophilic cysteine, a characteristic that has also been observed in the structurally similar low molecular weight tyrosine phosphatase.     

A Hybrid Mechanism for the Synechocystis Arsenate Reductase Revealed by Structural Snapshots during Arsenate Reduction

Evolution of enzymes plays a crucial role in obtaining new biological functions for all life forms. Arsenate reductases (ArsC) are several families of arsenic detoxification enzymes that reduce arsenate to arsenite, which can subsequently be extruded from cells by specific transporters. Among these, the Synechocystis ArsC (SynArsC) is structurally homologous to the well characterized thioredoxin (Trx)-coupled ArsC family but requires the glutaredoxin (Grx) system for its reactivation, therefore classified as a unique Trx/Grx-hybrid family. The detailed catalytic mechanism of SynArsC is unclear and how the “hybrid” mechanism evolved remains enigmatic. Herein, we report the molecular mechanism of SynArsC by biochemical and structural studies. Our work demonstrates that arsenate reduction is carried out via an intramolecular thiol-disulfide cascade similar to the Trx-coupled family, whereas the enzyme reactivation step is diverted to the coupling of the glutathione-Grx pathway due to the local structural difference. The current results support the hypothesis that SynArsC is likely a molecular fossil representing an intermediate stage during the evolution of the Trx-coupled ArsC family from the low molecular weight protein phosphotyrosine phosphatase (LMW-PTPase) family.     

Solution structures and backbone dynamics of arsenate reductase from Bacillus subtilis: reversible conformational switch associated with arsenate reduction

Arsenate reductase encoded by the chromosomal arsC gene in Bacillus subtilis catalyzes the intracellular reduction of arsenate to arsenite, which is then extruded from cells through an efficient and specific transport system. Herein, we present the solution structures and backbone dynamics of both the reduced and oxidized forms of arsenate reductase from B. subtilis. The overall structures of both forms are similar to those of bovine low molecular weight protein-tyrosine phosphatase and arsenate reductase from Staphylococcus aureus. However, several features of the tertiary structure and mobility are notably different between the reduced and oxidized forms of B. subtilis arsenate reductase, particularly in the P-loop region and the segment Cys(82)-Cys(89). The backbone dynamics results demonstrated that the reduced form of arsenate reductase undergoes millisecond conformational changes in the functional P-loop and Cys(82)-Cys(89), which may facilitate the formation of covalent intermediates and subsequent reduction of arsenate. In the oxidized form, Cys(82)-Cys(89) shows motional flexibility on both picosecond-to-nanosecond and possibly millisecond time scales, which may facilitate the reduction of the oxidized enzyme by thioredoxin to regenerate the active enzyme. Overall, the internal dynamics and static structures of the enzyme provide insights into the molecular mechanism of arsenate reduction, especially the reversible conformational switch and changes in internal motions associated with the catalytic reaction.     

Kinetics and active site dynamics of Staphylococcus aureus arsenate reductase

Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular arsenate(V) to the more toxic arsenite(III), which is subsequently extruded from the cell. It couples to thioredoxin, thioredoxin reductase and NADPH to be enzymatically active. ArsC is extremely sensitive to oxidative inactivation, has a very dynamic character hampering resonance assignments in NMR and produces peculiar biphasic Michaelis-Menten curves with two V(max) plateaus. In this study, methods to control ArsC oxidation during purification have been optimized. Next, application of Selwyn's test of enzyme inactivation was applied to progress curves and reveals that the addition of tetrahedral oxyanions (50 mM sulfate, phosphate or perchlorate) allows the control of ArsC stability and essentially eliminates the biphasic character of the Michaelis-Menten curves. Finally, 1H-15N HSQC NMR spectroscopy was used to establish that these oxyanions, including the arsenate substrate, exert their stabilizing effect on ArsC through binding with residues located within a C-X5-R sequence motif, characteristic for phosphotyrosine phosphatases. In view of this need for a tetrahedral oxyanion to structure its substrate binding site in its active conformation, a reappraisal of basic kinetic parameters of ArsC was necessary. Under these new conditions and in contrast to previous observations, ArsC has a high substrate specificity, as only arsenate could be reduced ( Km=68 microM, k(cat)/ Km =5.2 x 10(4 )M-1s-1), while its product, arsenite, was identified as a mixed inhibitor ( K*iu=534 microM, K*ic=377 microM).     

Mutagenesis study on amino acids around the molybdenum centre of the periplasmic nitrate reductase from Ralstonia eutropha

Molybdenum enzymes containing the pterin cofactor are a diverse group of enzymes that catalyse in general oxygen atom transfer reactions. Aiming at studying the amino acid residues, which are important for the enzymatic specificity, we used nitrate reductase from Ralstonia eutropha (R.e.NAP) as a model system for mutational studies at the active site. We mutated amino acids at the Mo active site (Cys181 and Arg421) as well as amino acids in the funnel leading to it (Met182, Asp196, Glu197, and the double mutant Glu197-Asp196). The mutations were made on the basis of the structural comparison of nitrate reductases with formate dehydrogenases (FDH), which show very similar three-dimensional structures, but clear differences in amino acids surrounding the active site. For mutations Arg421Lys and Glu197Ala we found a reduced nitrate activity while the other mutations resulted in complete loss of activity. In spite of the partial of total loss of nitrate reductase activity, these mutants do not, however, display FDH activity.     

Arsenate respiratory reductase gene (arrA) for Desulfosporosinus sp. strain Y5

Desulfosporosinus sp. strain Y5 is a spore-forming bacterium capable of dissimilatory arsenate reduction coupled to the oxidation of aromatic compounds. In arsenate respiration, the arsenate respiratory reductase (ARR) catalyzes the reduction of arsenate to arsenite. Our objective is to characterize the arrA gene, encoding the ARR, for Desulfosporosinus sp. strain Y5. Oligonucleotide primers were designed based on the few arrA gene sequences available at the time and validated against positive and negative controls. The resulting arrA-amplicon of approximately 2.0kb was cloned and sequenced. The arrA from Desulfosporosinus sp. Y5 is closely related to Desulfitobacterium hafniense (similarity of 77% and 81% at the nucleotide and amino acid levels, respectively). Phylogenetic topology based on the arrA gene was partially congruent with that of 16S rRNA-based analysis. This arrA sequence will support the development of specific tracking probes for Desulfosporosinus sp. Y5 and the molecular characterization and monitoring of dissimilatory arsenate reducing bacteria.     

Ferredoxin-NADP+ reductase from Plasmodium falciparum undergoes NADP+-dependent dimerization and inactivation: functional and crystallographic analysis

The completion of the Plasmodium falciparum genome sequence has recently promoted the search for new antimalarial drugs. More specifically, metabolic pathways of the apicoplast, a key organelle for survival of the parasite, have been recognized as potential targets for the development of specific new antimalarial agents. As most apicomplexan parasites, P. falciparum displays a plant-type ferredoxin-NADP(+) reductase, yielding reduced ferredoxin for essential biosynthetic pathways in the apicoplast. Here we report a molecular, kinetic and ligand binding characterization of the recombinant ferredoxin-NADP(+) reductase from P. falciparum, in the light of current data available for plant ferredoxin-NADP(+) reductases. In parallel with the functional characterization, we describe the crystal structures of P. falciparum ferredoxin-NADP(+) reductase in free form and in complex with 2'-phospho-AMP (at 2.4 and 2.7 A resolution, respectively). The enzyme displays structural properties likely to be unique to plasmodial reductases. In particular, the two crystal structures highlight a covalent dimer, which relies on the oxidation of residue Cys99 in two opposing subunits, and a helix-coil transition that occurs in the NADP-binding domain, triggered by 2'-phospho-AMP binding. Studies in solution show that NADP(+), as well as 2'-phospho-AMP, promotes the formation of the disulfide-stabilized dimer. The isolated dimer is essentially inactive, but full activity is recovered upon disulfide reduction. The occurrence of residues unique to the plasmodial enzyme, and the discovery of specific conformational properties, highlight the NADP-binding domain of P. falciparum ferredoxin-NADP(+) reductase as particularly suited for the rational development of antimalarial compounds.     

Development of a viologen-based microtiter plate assay for the analysis of oxyanion reductase activity: application to the membrane-bound selenate reductase from Enterobacter cloacae SLD1a-1

The membrane-bound selenate reductase of Enterobacter cloacae SLD1a-1 is purified in low yield and has relatively low activity in the pure form compared to that of other oxyanion reductases, such as the membrane-bound and periplasmic nitrate reductases. A microtiter plate assay based on the original quartz cuvette viologen assay of Jones and Garland (R.W. Jones, P.B. Garland, Biochem. J 164 (1977) 199-211) was developed specifically for analysis of such low-abundant, labile oxyanion reductases. The plate assay detects the enzyme-dependent reoxidation of reduced methyl viologen spectrophotometrically at 600 nm. The assay is quick, uses a minimal sample volume (<5 microl), can simultaneously test a range of alternative substrates, and permits activity measurements on multiple samples. We demonstrate the accuracy and versatility of the microtiter plate assay by application to the kinetic analysis, inhibition, and pH optimization of the membrane-bound selenate reductase from E. cloacae SLD1a-1. Results show that the membrane-bound selenate reductase has optimum activity at pH approximately 8 and its active site is able to accommodate larger inhibitory complexes resulting in mixed-type inhibition, in the presence of selenate and potassium thiocyanate.     

Redox, mutagenic and structural studies of the glutaredoxin/arsenate reductase couple from the cyanobacterium Synechocystis sp. PCC 6803

The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation-reduction midpoint potential (E_m) value of − 165 mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E_m value of − 220 mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8 Å resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme.     

Structure and mutation analysis of archaeal geranylgeranyl reductase

The crystal structure of geranylgeranyl reductase (GGR) from Sulfolobus acidocaldarius was determined in order to elucidate the molecular mechanism of the catalytic reaction. The enzyme is a flavoprotein and is involved in saturation of the double bonds on the isoprenoid moiety of archaeal membranes. The structure determined in this study belongs to the p-hydroxybenzoate hydroxylase family in the glutathione reductase superfamily. GGR functions as a monomer and is divided into the FAD-binding, catalytic and C-terminal domains. The catalytic domain has a large cavity surrounded by a characteristic YxWxFPx_7-8GxG motif and by the isoalloxazine ring of an FAD molecule. The cavity holds a lipid molecule, which is probably derived from Escherichia coli cells used for over-expression. One of the two forms of the structure clarifies the presence of an anion pocket holding a pyrophosphate molecule, which might anchor the phosphate head of the natural ligands. Mutational analysis supports the suggestion that the three aromatic residues of the YxWxFPx_7-8GxG motif hold the ligand in the appropriate position for reduction. Cys47, which is widely conserved in GGRs, is located at the si-side of the isoalloxazine ring of FAD and is shown by mutational analysis to be involved in catalysis. The catalytic cycle, including the FAD reducing factor binding site, is proposed on the basis of the detailed analysis of the structure.     

Characterization of mitochondrial thioredoxin reductase from C. elegans

Thioredoxin reductase catalyzes the NADPH-dependent reduction of the catalytic disulfide bond of thioredoxin. In mammals and other higher eukaryotes, thioredoxin reductases contain the rare amino acid selenocysteine at the active site. The mitochondrial enzyme from Caenorhabditis elegans, however, contains a cysteine residue in place of selenocysteine. The mitochondrial C. elegans thioredoxin reductase was cloned from an expressed sequence tag and then produced in Escherichia coli as an intein-fusion protein. The purified recombinant enzyme has a kcat of 610 min(-1) and a Km of 610 microM using E. coli thioredoxin as substrate. The reported kcat is 25% of the kcat of the mammalian enzyme and is 43-fold higher than a cysteine mutant of mammalian thioredoxin reductase. The enzyme would reduce selenocysteine, but not hydrogen peroxide or insulin. The flanking glycine residues of the GCCG motif were mutated to serine. The mutants improved substrate binding, but decreased the catalytic rate.     

Crystal structure of Saccharomyces cerevisiae 3'-phosphoadenosine-5'-phosphosulfate reductase complexed with adenosine 3',5'-bisphosphate

Most assimilatory bacteria, fungi, and plants species reduce sulfate (in the activated form of APS or PAPS) to produce reduced sulfur. In yeast, PAPS reductase reduces PAPS to sulfite and PAP. Despite the difference in substrate specificity and catalytic cofactor, PAPS reductase is homologous to APS reductase in both sequence and structure, and they are suggested to share the same catalytic mechanism. Metazoans do not possess the sulfate reduction pathway, which makes APS/PAPS reductases potential drug targets for human pathogens. Here, we present the 2.05 A resolution crystal structure of the yeast PAPS reductase binary complex with product PAP bound. The N-terminal region mediates dimeric interactions resulting in a unique homodimer assembly not seen in previous APS/PAPS reductase structures. The "pyrophosphate-binding" sequence (47)TTAFGLTG(54) defines the substrate 3'-phosphate binding pocket. In yeast, Gly54 replaces a conserved aspartate found in APS reductases vacating space and charge to accommodate the 3'-phosphate of PAPS, thus regulating substrate specificity. Also, for the first time, the complete C-terminal catalytic motif (244)ECGIH(248) is revealed in the active site. The catalytic residue Cys245 is ideally positioned for an in-line attack on the beta-sulfate of PAPS. In addition, the side chain of His248 is only 4.2 A from the Sgamma of Cys245 and may serve as a catalytic base to deprotonate the active site cysteine. A hydrophobic sequence (252)RFAQFL(257) at the end of the C-terminus may provide anchoring interactions preventing the tail from swinging away from the active site as seen in other APS/PAPS reductases.     

Crystal structure of the soluble domain of the major anaerobically induced outer membrane protein (AniA) from pathogenic Neisseria: a new class of copper-containing nitrite reductases

The major anaerobically induced outer membrane protein (AniA) from pathogenic Neisseria gonorrhoeae is essential for cell growth under oxygen limiting conditions in the presence of nitrite and is protective against killing by human sera. A phylogenic analysis indicates that AniA is a member of a new class of copper-containing nitrite reductases. Expression of the soluble domain of AniA yields a protein capable of reducing nitrite with specific activity of 160 units/mg, approximately 50 % of that measured for the nitrite reductase from the strong soil denitrifier Alcaligenes faecalis S-6. The crystal structure of the soluble domain of AniA was solved by molecular replacement and sixfold averaging to a resolution of 2.4 A. The nitrite soaked AniA crystal structure refined to 1.95 A reveals a bidentate mode of substrate binding to the type II copper. Despite low sequence identity (approximately 30 %), the core cupredoxin fold of AniA is similar to that found in copper-containing nitrite reductases from soil bacteria. The main structural differences are localized to two attenuated surface loops that map to deletions in the sequence alignment. In soil nitrite reductases, one of these surface loops is positioned near the type I copper site and contributes residues to the docking surface for proteaceous electron donors. In AniA, the attenuation of this loop results in a restructured hydrophobic binding surface that may be required to interact with a lipid anchored azurin. The second attenuated loop is positioned on the opposite side of AniA and may facilitate a more intimate interaction with the lipid membrane. A unique combination of structural effectors surrounding the type I copper site of sAnia contribute to a unusual visible absorption spectra with components observed previously in either green or blue type I copper sites.     

The crystal structure of the bifunctional deaminase/reductase RibD of the riboflavin biosynthetic pathway in Escherichia coli: implications for the reductive mechanism

We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1-145) is similar to cytosine deaminase and the C-terminal domain (146-367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP(+)) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159-173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.     

A structural analysis of the catalytic mechanism of methionine sulfoxide reductase A from Neisseria meningitidis

The methionine sulfoxide reductases (Msrs) are thioredoxin-dependent oxidoreductases that catalyse the reduction of the sulfoxide function of the oxidized methionine residues. These enzymes have been shown to regulate the life span of a wide range of microbial and animal species and to play the role of physiological virulence determinant of some bacterial pathogens. Two structurally unrelated classes of Msrs exist, MsrA and MsrB, with opposite stereoselectivity towards the R and S isomers of the sulfoxide function, respectively. Both Msrs share a similar three-step chemical mechanism including (1) the formation of a sulfenic acid intermediate on the catalytic Cys with the concomitant release of the product—methionine, (2) the formation of an intramonomeric disulfide bridge between the catalytic and the regenerating Cys and (3) the reduction of the disulfide bridge by thioredoxin or its homologues. In this study, four structures of the MsrA domain of the PilB protein from Neisseria meningitidis, representative of four catalytic intermediates of the MsrA catalytic cycle, were determined by X-ray crystallography: the free reduced form, the Michaelis-like complex, the sulfenic acid intermediate and the disulfide oxidized forms. They reveal a conserved overall structure up to the formation of the sulfenic acid intermediate, while a large conformational switch is observed in the oxidized form. The results are discussed in relation to those proposed from enzymatic, NMR and theoretical chemistry studies. In particular, the substrate specificity and binding, the catalytic scenario of the reductase step and the relevance and role of the large conformational change observed in the oxidized form are discussed.     

Fine tuning of coenzyme specificity in family 2 aldo-keto reductases revealed by crystal structures of the Lys-274-->Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+

Aldo-keto reductases of family 2 employ single site replacement Lys-->Arg to switch their cosubstrate preference from NADPH to NADH. X-ray crystal structures of Lys-274-->Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+ were determined at a resolution of 2.4 and 2.3A, respectively. Due to steric conflicts in the NADP+-bound form, the arginine side chain must rotate away from the position of the original lysine side chain, thereby disrupting a network of direct and water-mediated interactions between Glu-227, Lys-274 and the cofactor 2'-phosphate and 3'-hydroxy groups. Because anchoring contacts of its Glu-227 are lost, the coenzyme-enfolding loop that becomes ordered upon binding of NAD(P)+ in the wild-type remains partly disordered in the NADP+-bound mutant. The results delineate a catalytic reaction profile for the mutant in comparison to wild-type.