Internal Aldimine Research Articles

Mechanism-based Inhibition Reveals Transitions between Two Conformational States in the Action of Lysine 5,6-Aminomutase: A Combination of Electron Paramagnetic Resonance Spectroscopy, Electron Nuclear Double Resonance Spectroscopy, and Density Functional Theory Study

An "open"-state crystal structure of lysine 5,6-aminomutase suggests that transition to a hypothetical "closed"-state is required to bring the cofactors adenosylcobalamin (AdoCbl) and pyridoxal-5'-phosphate (PLP) and the substrate into proximity for the radical-mediated 1,2-amino group migration. This process is achieved by transaldimination of the PLP-Lys144β internal aldimine with the PLP-substrate external aldimine. A closed-state crystal structure is not available. UV-vis and electron paramagnetic resonance studies show that homologues of substrate D-lysine, 2,5-DAPn, 2,4-DAB, and 2,3-DAPr bind to PLP as an external aldimine and elicit the AdoCbl Co-C bond homolysis and the accumulations of cob(II)alamin and analogue-based radicals, demonstrating the existence of a closed state. (2)H- and (31)P-electron nuclear double resonance studies, supported by computations, show that the position for hydrogen atom abstraction from 2,5-DAPn and 2,4-DAB by the 5'-deoxyadenosyl radical occurs at the carbon adjacent to the imine, resulting in overstabilized radicals by spin delocalization through the imine into the pyridine ring of PLP. These radicals block the active site, inhibit the enzyme, and poise the enzyme into two distinct conformations: for even-numbered analogues, the cob(II)alamin remains proximal to and spin-coupled with the analogue-based radical in the closed state while odd-numbered analogues could trigger the transition to the open state of the enzyme. We provide here direct spectroscopic evidence that strongly support the existence of a closed state and its analogue-dependent transition to the open state, which is one step that was proposed to complete the catalytic turnover of the substrate lysine.

Detection of Reaction Intermediates during Human Cystathionine β-Synthase-monitored Turnover and H2S Production

Human cystathionine β-synthase (CBS), a novel heme-containing pyridoxal 5'-phosphate enzyme, catalyzes the condensation of homocysteine and serine or cysteine to produce cystathionine and H(2)O or H(2)S, respectively. The presence of heme in CBS has limited spectrophotometric characterization of reaction intermediates by masking the absorption of the pyridoxal 5'-phosphate cofactor. In this study, we employed difference stopped-flow spectroscopy to characterize reaction intermediates formed under catalytic turnover conditions. The reactions of L-serine and L-cysteine with CBS resulted in the formation of a common aminoacrylate intermediate (k(obs) = 0.96 ± 0.02 and 0.38 ± 0.01 mM(-1) s(-1), respectively, at 24 °C) with concomitant loss of H(2)O and H(2)S and without detectable accumulation of the external aldimine or other intermediates. Homocysteine reacted with the aminoacrylate intermediate with k(obs) = 40.6 ± 3.8 s(-1) and re-formed the internal aldimine. In the reverse direction, CBS reacted with cystathionine, forming the aminoacrylate intermediate with k(obs) = 0.38 ± 0.01 mM(-1) s(-1). This study provides the first insights into the pre-steady-state kinetic mechanism of human CBS and indicates that the reaction is likely to be limited by a conformational change leading to product release.

Structures of a γ-aminobutyrate (GABA) transaminase from the s-triazine-degrading organism Arthrobacter aurescens TC1 in complex with PLP and with its external aldimine PLP-GABA adduct.

Two complex structures of the γ-aminobutyrate (GABA) transaminase A1R958 from Arthrobacter aurescens TC1 are presented. The first, determined to a resolution of 2.80 Å, features the internal aldimine formed by reaction between the ℇ-amino group of Lys295 and the cofactor pyridoxal phosphate (PLP); the second, determined to a resolution of 2.75 Å, features the external aldimine adduct formed between PLP and GABA in the first half-reaction. This is the first structure of a microbial GABA transaminase in complex with its natural external aldimine and reveals the molecular determinants of GABA binding in this enzyme.

Structural insights into catalysis by βC‐S lyase from Streptococcus anginosus

Hydrogen sulfide (H(2)S) is a causative agent of oral malodor and may play an important role in the pathogenicity of oral bacteria such as Streptococcus anginosus. In this microorganism, H(2)S production is associated with βC-S lyase (Lcd) encoded by lcd gene, which is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the α,β-elimination of sulfur-containing amino acids. When Lcd acts on L-cysteine, H(2)S is produced along with pyruvate and ammonia. To understand the H(2)S-producing mechanism of Lcd in detail, we determined the crystal structures of substrate-free Lcd (internal aldimine form) and two reaction intermediate complexes (external aldimine and α-aminoacrylate forms). The formation of intermediates induced little changes in the overall structure of the enzyme and in the active site residues, with the exception of Lys234, a PLP-binding residue. Structural and mutational analyses highlighted the importance of the active site residues Tyr60, Tyr119, and Arg365. In particular, Tyr119 forms a hydrogen bond with the side chain oxygen atom of L-serine, a substrate analog, in the external aldimine form suggesting its role in the recognition of the sulfur atom of the true substrate (L-cysteine). Tyr119 also plays a role in fixing the PLP cofactor at the proper position during catalysis through binding with its side chain. Finally, we partly modified the catalytic mechanism known for cystalysin, a βC-S lyase from Treponema denticola, and proposed an improved mechanism, which seems to be common to the βC-S lyases from oral bacteria.

Pharmacokinetics, immunogenicity and anticancer efficiency of Aspergillus flavipesl-methioninase

Methionine starvation can powerfully modulate DNA methylation, cell cycle transition, polyamines and antioxidant synthesis of tumor cells, in contrary to normal ones. Aspergillus flavipesl-methioninase was previously characterized by our studies, displaying affordable biochemical properties comparing to Pseudomonas putida enzyme (ONCASE). Thus, the objective of current study was to evaluate the catalytic properties of Af-METase in New Zealand rabbits, exploring its antitumor efficacy. In vivo, Af-METase (40.8U/ml) have T1/2 19.8h, elimination constant 0.088U/h and apparent volume distribution 85U/ml. Also, Af-METase has two maxima one at A280nm (apo-enzyme) and at A420nm (internal Schiff base of PLP), unlike control plasma (without enzyme). The two peaks of absorption spectra were detected maximally at 15min then the absorbance at 420nm was subsequently decreased with circulation time, due to dissociation of the co-enzyme. The A280/420 ratio was increased from 1.69 to 5.81 with circulation time from 15 to 30h. Rabbits plasma methionine was depleted from 18.7μM (control) to 8.8μM after 1h of enzyme injection and completely omitted after 2h till 19h, assuming the sustainability of negligible levels of methionine (<2μM) in plasma of rabbits, for about 17h. Upon infusion of PLP, the T1/2 of Af-METase was significantly prolonged by 3.2 fold, assuming the fully reconstitution of the enzyme. The holo-AfMETase still retained its co-enzyme, completely, till 33h of PLP infusion. From spectral studies, the internal aldimine linkage of apo-Af-METase was constructed upon PLP infusion, with fully catalytic structure after less than 4h of its infusion, the A280/420 ratio being not relatively changed till 45h. After 25 days of last enzyme dose, the titer of IgG was increase by about 1.66 fold comparing to control (without enzyme). However, IgM was not detected along the tested challenge points. In vitro, plasma anti-Af-METase neutralizing antibodies (NAb) were assessed, with no significant reduction on activity of Af-METase by Nab. All the hematological parameters were in normal range, otherwise, the RBCs titer and platelet level was slightly increased, after 25 days of Af-METase injection, comparing to control. There is no obvious negative effect on chemistry of liver, kidney, glucose, lipids, and other electrolytes. Additionally, the anticancer activity of Af-METase was evaluated against five types of human cancer cell lines, in vitro. The enzyme showed a powerful activity against prostate (PC3), liver (HEPG2) and breast (MCF7) cancers, with IC50 0.001U/ml, 0.26U/ml and 0.37U/ml, respectively.

Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acid α-decarboxylases

Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to β-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development.

Crystal structure and substrate specificity of the thermophilic serine:pyruvate aminotransferase fromSulfolobus solfataricus

The three-dimensional structure of the Sulfolobus solfataricus serine:pyruvate aminotransferase has been determined to 1.8 Å resolution. The structure of the protein is a homodimer that adopts the type I fold of pyridoxal 5'-phosphate (PLP)-dependent aminotransferases. The structure revealed the PLP cofactor covalently bound in the active site to the active-site lysine in the internal aldimine form. The structure of the S.solfataricus enzyme was also determined with an amino form of the cofactor pyridoxamine 5'-phosphate bound in the active site and in complex with gabaculine, an aminotransferase inhibitor. These structures showed the changes in the enzyme active site during the course of the catalytic reaction. A comparison of the structure of the S. solfataricus enzyme with that of the closely related alanine:glyoxylate aminotransferase has identified structural features that are proposed to be responsible for the differences in substrate specificity between the two enzymes. These results have been complemented by biochemical studies of the substrate specificity and thermostability of the S. solfataricus enzyme.

Crystal Structure of Citrobacter freundii Asp214Ala Tyrosine Phenollyase Reveals that Asp214 is Critical for Maintaining a Strain in the Internal Aldimine

Tyrosine phenol-lyase (TPL) is a pyridoxal-5′-phosphate (PLP) dependent enzyme which catalyzes β-elimination of L-tyrosine. In the holoenzyme the protonated pyridinium N1 atom of the PLP cofactor is hydrogen-bonded to the side chain of Asp214. Here we report the X-ray structure of C. freundii D214A TPL determined at 1.9 A resolution. Comparison with the structure of the wild-type TPL shows that the D214A replacement induced significant conformational reorganization in the active site leading to its partial closure. Significantly, in D214A TPL the strain in the internal aldimine is completely released and the pyridine N1 atom of PLP is deprotonated. These observations explain the considerably reduced activity of the D214A TPL towards its substrates [T. V. Demidkina et al., Biochim. Biophys. Acta, Proteins Proteomics 1764 (2006) 1268–1276]. The reported structure reveals that Asp214 is critical for maintaining the strain in the internal aldimine. We argue that this strain is used by the enzyme to accelerate the transaldimination reaction, the first step in the enzymatic catalysis.

Mechanism of the aromatic aminotransferase encoded by the Aro8 gene from Saccharomyces cerevisiae

The amino acid l-lysine is synthesized in Saccharomyces cerevisiae via the α-aminoadipate pathway. An as yet unidentified PLP-containing aminotransferase is thought to catalyze the formation of α-aminoadipate from α-ketoadipate in the l-lysine biosynthetic pathway that could be the yeast Aro8 gene product. A screen of several different amino acids and keto-acids showed that the enzyme uses l-tyrosine, l-phenylalanine, α-ketoadipate, and l-α-aminoadipate as substrates. The UV–visible spectrum of the aminotransferase exhibits maxima at 280 and 343 nm at pH 7.5. As the pH is decreased the peak at 343 nm (the unprotonated internal aldimine) disappears and two new peaks at 328 and 400 nm are observed representing the enolimine and ketoenamine tautomers of the protonated aldimine, respectively. Addition, at pH 7.1, of α-ketoadipate to free enzyme leads to disappearance of the absorbance at 343 nm and appearance of peaks at 328 and 424 nm. The V/ E t and V/K α-ketoadipate E t pH profiles are pH independent from pH 6.5 to 9.6, while the V/K l -tyrosine pH-rate profile decreases below a single p K a of 7.0 ± 0.1. Data suggest the active enzyme form is with the internal aldimine unprotonated. We conclude the enzyme should be categorized as a α-aminoadipate aminotransferase.

Mechanism of Formation of the Internal Aldimine in Pyridoxal 5′-Phosphate-Dependent Enzymes

In this paper we studied the mechanism of formation of the internal aldimine, a common intermediate to most pyridoxal 5'-phosphate (PLP)-dependent enzymes. A large model based on the crystal structure from the human ornithine decarboxylase (ODC) enzyme was constructed and in total accounts for 504 atoms. The reaction mechanism was investigated using the ONIOM methodology (B3LYP/6-31G(d)//AM1), and the final energies were calculated with the M06/6-311++G(2d,2p)//B3LYP/6-31G(d) level of theory. It was demonstrated that the reaction is accomplished in three sequential steps: (i) the nucleophilic attack of Lysine69 to PLP, (ii) the carbinolamine formation, and (iii) a final dehydration step. For the carbinolamine formation, several mechanistic hypotheses were explored, and the preferred pathway assigns a key role for the conserved active site Cys360. The overall reaction is exergonic in -9.1 kcal/mol, and the rate-limiting step is the dehydration step (E(a) = 13.5 kcal/mol). For the first time, we provide an atomistic portrait of this mechanism in an enzymatic environment. Moreover, we were able to assign a novel role to Cys360 in the ODC reaction mechanism that was never proposed.

Theoretical and X‐ray Crystallographic Evidence of a Fluorine‐Imine Gauche Effect: An Addendum to Dunathan’s Stereoelectronic Hypothesis

The preference of β-fluoroimines to adopt a gauche conformation has been studied by single-crystal X-ray diffraction analysis and DFT methods. Empirical and theoretical evidence for a preferential gauche arrangement around the NCCF torsion angle (φ) is presented ((E)-2-fluoro-N-(4-nitrobenzylidene)ethanamine: φ(NCCF) =70.0°). In the context of this study, the analysis of a pyridoxal-derived β-fluoroaldimine was performed, a species that is implicated in the inhibition of pyridoxal phosphate (PLP)-dependent enzymes by β-fluoroamine derivatives. The gauche preference of the internal aldimine (=NCH(2)CH(2)F) that can be rationalized by stereoelectronic arguments does not hold for the corresponding external system (N=CHCH(2)F) (E(min) when φ(NCCF) =120°). Moreover, the C-F bond is lengthened by more than 0.02 Å at φ(NCCF) =±90°, when it is exactly antiperiplanar to the conjugated imine. This activation of the C-F σ bond by an adjacent π system constitutes an addendum to Dunathan's stereoelectronic hypothesis.

Crystal Structures of Aspartate Aminotransferase Reconstituted with 1-Deazapyridoxal 5′-Phosphate: Internal Aldimine and Stable l-Aspartate External Aldimine

The 1.8 Å resolution crystal structures of Escherichia coli aspartate aminotransferase reconstituted with 1-deazapyridoxal 5'-phosphate (deazaPLP; 2-formyl-3-hydroxy-4-methylbenzyl phosphate) in the internal aldimine and L-aspartate external aldimine forms are reported. The L-aspartate·deazaPLP external aldimine is extraordinarily stable (half-life of >20 days), allowing crystals of this intermediate to be grown by cocrystallization with L-aspartate. This structure is compared to that of the α-methyl-L-aspartate·PLP external aldimine. Overlays with the corresponding pyridoxal 5'-phosphate (PLP) aldimines show very similar orientations of deazaPLP with respect to PLP. The lack of a hydrogen bond between Asp222 and deazaPLP, which serves to "anchor" PLP in the active site, releases strain in the deazaPLP internal aldimine that is enforced in the PLP internal aldimine [Hayashi, H., Mizuguchi, H., Miyahara, I., Islam, M. M., Ikushiro, H., Nakajima, Y., Hirotsu, K., and Kagamiyama, H. (2003) Biochim. Biophys. Acta1647, 103] as evidenced by the planarity of the pyridine ring and the Schiff base linkage with Lys258. Additionally, loss of this anchor causes a 10° greater tilt of deazaPLP toward the substrate in the external aldimine. An important mechanistic difference between the L-aspartate·deazaPLP and α-methyl-L-aspartate·PLP external aldimines is a hydrogen bond between Gly38 and Lys258 in the former, positioning the catalytic base above and approximately equidistant between Cα and C4'. In contrast, in the α-methyl-L-aspartate·PLP external aldimine, the ε-amino group of Lys258 is rotated ~70° to form a hydrogen bond to Tyr70 because of the steric bulk of the methyl group.

Computational Mechanistic Studies Addressed to the Transimination Reaction Present in All Pyridoxal 5'-Phosphate-Requiring Enzymes.

The pyridoxal-5'-phosphate-dependent enzymes (PLP enzymes) catalyze a myriad of biochemical reactions, being actively involved in the biosynthesis of amino acids and amino acid-derived metabolites as well as in the biosynthetic pathways of amino sugars and in the synthesis or catabolism of neurotransmitters. Although the scope of PLP-catalyzed reactions initially appears to be bewilderingly diverse, there is a simple unifying principle: In the resting state, the cofactor (PLP) is covalently bonded to the amino group of an active site lysine, forming an internal aldimine. Once the amino substrate interacts with the active site, a new Schiff base is generated, commonly referred to as the external aldimine. Only after this step, the mechanistic pathway for each PLP-catalyzed reaction diverges. In this paper, density functional methods have been applied to investigate this common step present in all PLP-dependent enzymes-the transimination reaction. The results indicate that the reaction involves three sequential steps: (i) formation of a tetrahedral intermediate with the active site lysine and the amino substrate bonded to the PLP cofactor; (ii) nondirect proton transfer between the amino substrate and the lysine residue; and (iii) formation of the external aldimine after the dissociation of the lysine residue. The overall reaction is exothermic (-12.0 kcal/mol), and the rate-limiting step is the second one with 12.6 kcal/mol for the activation energy.

Product-assisted Catalysis as the Basis of the Reaction Specificity of Threonine Synthase

Threonine synthase (TS), which is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the elimination of the γ-phosphate group from O-phospho-L-homoserine (OPHS) and the subsequent addition of water at Cβ to form L-threonine. The catalytic course of TS is the most complex among the PLP enzymes, and it is an intriguing problem how the elementary steps are controlled in TS to carry out selective reactions. When L-vinylglycine was added to Thermus thermophilus HB8 TS in the presence of phosphate, L-threonine was formed with k(cat) and reaction specificity comparable with those when OPHS was used as the substrate. However, in the absence of phosphate or when sulfate was used in place of phosphate, only the side reaction product, α-ketobutyrate, was formed. Global analysis of the spectral changes in the reaction of TS with L-threonine showed that compared with the more acidic sulfate ion, the phosphate ion decreased the energy levels of the transition states of the addition of water at the Cβ of the PLP-α-aminocrotonate aldimine (AC) and the transaldimination to form L-threonine. The x-ray crystallographic analysis of TS complexed with an analog for AC gave a distinct electron density assigned to the phosphate ion derived from the solvent near the Cβ of the analog. These results indicated that the phosphate ion released from OPHS by γ-elimination acts as the base catalyst for the addition of water at Cβ of AC, thereby providing the basis of the reaction specificity. The phosphate ion is also considered to accelerate the protonation/deprotonation at Cγ.

Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine β-synthase

The catalytic potential for H(2)S biogenesis and homocysteine clearance converge at the active site of cystathionine β-synthase (CBS), a pyridoxal phosphate-dependent enzyme. CBS catalyzes β-replacement reactions of either serine or cysteine by homocysteine to give cystathionine and water or H(2)S, respectively. In this study, high-resolution structures of the full-length enzyme from Drosophila in which a carbanion (1.70 Å) and an aminoacrylate intermediate (1.55 Å) have been captured are reported. Electrostatic stabilization of the zwitterionic carbanion intermediate is afforded by the close positioning of an active site lysine residue that is initially used for Schiff base formation in the internal aldimine and later as a general base. Additional stabilizing interactions between active site residues and the catalytic intermediates are observed. Furthermore, the structure of the regulatory "energy-sensing" CBS domains, named after this protein, suggests a mechanism for allosteric activation by S-adenosylmethionine.

Structural Analysis of WbpE from Pseudomonas aeruginosa PAO1: A Nucleotide Sugar Aminotransferase Involved in O-Antigen Assembly,

In recent years, the opportunistic pathogen Pseudomonas aeruginosa has emerged as a major source of hospital-acquired infections. Effective treatment has proven increasingly difficult due to the spread of multidrug resistant strains and thus requires a deeper understanding of the biochemical mechanisms of pathogenicity. The central carbohydrate of the P. aeruginosa PAO1 (O5) B-band O-antigen, ManNAc(3NAc)A, has been shown to be critical for virulence and is produced in a stepwise manner by five enzymes in the Wbp pathway (WbpA, WbpB, WbpE, WbpD, and WbpI). Herein, we present the crystal structure of the aminotransferase WbpE from P. aeruginosa PAO1 in complex with the cofactor pyridoxal 5'-phosphate (PLP) and product UDP-GlcNAc(3NH(2))A as the external aldimine at 1.9 A resolution. We also report the structures of WbpE in complex with PMP alone as well as the PLP internal aldimine and show that the dimeric structure of WbpE observed in the crystal structure is confirmed by analytical ultracentrifugation. Analysis of these structures reveals that the active site of the enzyme is composed of residues from both subunits. In particular, we show that a key residue (Arg229), which has previously been implicated in direct interactions with the alpha-carboxylate moiety of alpha-ketoglutarate, is also uniquely positioned to bestow specificity for the 6''-carboxyl group of GlcNAc(3NH(2))A through a salt bridge. This finding is intriguing because while an analogous basic residue is present in WbpE homologues that do not process 6''-carboxyl-modified saccharides, recent structural studies reveal that this side chain is retracted to accommodate a neutral C6'' atom. This work represents the first structural analysis of a nucleotide sugar aminotransferase with a bound product modified at the C2'', C3'', and C6'' positions and provides insight into a novel target for treatment of P. aeruginosa infection.

Insights into the Mechanism ofPseudomonas dacunhaeAspartate β-Decarboxylase from Rapid-Scanning Stopped-Flow Kinetics

The mechanism of wild-type and R37A mutant Pseudomonas dacunhae aspartate beta-decarboxylase (ABDC) was studied by rapid-scanning stopped-flow spectrophotometry. Mixing wild-type ABDC with 50 mM disodium l-Asp resulted in the formation of a 325 nm absorption peak within the dead time of the stopped-flow instrument, likely the ketimine of pyridoxamine 5'-phosphate and oxaloacetate or pyruvate. After consumption of the l-Asp, the 360 nm feature of the resting enzyme was restored. Thus, the 325 nm species is a catalytically competent intermediate. In contrast, mixing wild-type ABDC with the disodium salt of either threo- or erythro-beta-hydroxy-dl-Asp at 50 mM resulted in a much slower formation of the 325 nm complex, with an apparent rate constant of approximately 1 or 0.006 s(-1), respectively. When wild-type ABDC is mixed with disodium succinate, a nonreactive analogue of l-Asp, formation of a new peak at 425 nm is observed. The apparent rate constant for formation of the 425 nm band exhibits a hyperbolic dependence on succinate concentration, showing that there is a rapid binding equilibrium, followed by a slower reaction in which the internal aldimine is protonated on the Schiff base N. Hydrostatic pressure shifts the spectrum from the 425 nm form to the 360 nm form, consistent with a conformational change. It is likely that the binding of substrate or analogues induces a conformational change that releases strain in the Lys pyridoxal 5'-phosphate Schiff base and increases the pK(a), resulting in protonation of the Schiff base to initiate transaldimination. Mixing of R37A mutant ABDC with 50 mM l-Asp also results in the formation of the 325 nm complex, but with an apparent rate constant of 0.2 s(-1), at least 5000-fold slower than the rate of wild-type ABDC. In contrast to wild-type ABDC, R37A ABDC shows no change in the cofactor spectrum when mixed with disodium succinate. These results suggest that Arg-37, a conserved active site residue in ABDC, plays a role in modulating the pK(a) of the pyridoxal 5'-phosphate complexes during catalysis.

Large-scale Domain Dynamics and Adenosylcobalamin Reorientation Orchestrate Radical Catalysis in Ornithine 4,5-Aminomutase

D-ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii converts D-ornithine to 2,4-diaminopentanoic acid by way of radical propagation from an adenosylcobalamin (AdoCbl) to a pyridoxal 5'-phosphate (PLP) cofactor. We have solved OAM crystal structures in different catalytic states that together demonstrate unusual stability of the AdoCbl Co-C bond and that radical catalysis is coupled to large-scale domain motion. The 2.0-A substrate-free enzyme crystal structure reveals the Rossmann domain, harboring the intact AdoCbl cofactor, is tilted toward the edge of the PLP binding triose-phosphate isomerase barrel domain. The PLP forms an internal aldimine link to the Rossmann domain through Lys(629), effectively locking the enzyme in this "open" pre-catalytic conformation. The distance between PLP and 5'-deoxyadenosyl group is 23 A, and large-scale domain movement is thus required prior to radical catalysis. The OAM crystals contain two Rossmann domains within the asymmetric unit that are unconstrained by the crystal lattice. Surprisingly, the binding of various ligands to OAM crystals (in an oxygen-free environment) leads to transimination in the absence of significant reorientation of the Rossmann domains. In contrast, when performed under aerobic conditions, this leads to extreme disorder in the latter domains correlated with the loss of the 5'-deoxyadenosyl group. Our data indicate turnover and hence formation of the "closed" conformation is occurring within OAM crystals, but that the equilibrium is poised toward the open conformation. We propose that substrate binding induces large-scale domain motion concomitant with a reconfiguration of the 5'-deoxyadenosyl group, triggering radical catalysis in OAM.

Reaction Mechanism and Molecular Basis for Selenium/Sulfur Discrimination of Selenocysteine Lyase

Selenocysteine lyase (SCL) catalyzes the pyridoxal 5'-phosphate-dependent removal of selenium from l-selenocysteine to yield l-alanine. The enzyme is proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residue as an essential component. The enzyme exhibits strict substrate specificity toward l-selenocysteine and no activity to its cognate l-cysteine. However, it remains unclear how the enzyme distinguishes between selenocysteine and cysteine. Here, we present mechanistic studies of selenocysteine lyase from rat. ESI-MS analysis of wild-type and C375A mutant SCL revealed that the catalytic reaction proceeds via the formation of an enzyme-bound selenopersulfide intermediate on the catalytically essential Cys-375 residue. UV-visible spectrum analysis and the crystal structure of SCL complexed with l-cysteine demonstrated that the enzyme reversibly forms a nonproductive adduct with l-cysteine. Cys-375 on the flexible loop directed l-selenocysteine, but not l-cysteine, to the correct position and orientation in the active site to initiate the catalytic reaction. These findings provide, for the first time, the basis for understanding how trace amounts of a selenium-containing substrate is distinguished from excessive amounts of its cognate sulfur-containing compound in a biological system.

Kinetic Analysis of Cysteine Desulfurase CD0387 from Synechocystis sp. PCC 6803: Formation of the Persulfide Intermediate

Stopped-flow absorption and isotope effect experiments have been used to dissect the mechanism of formation of the enzyme cysteinyl persulfide intermediate in the reaction of a cysteine desulfurase (CD), CD0387 from Synechocystis sp. strain PCC 6803. Seven accumulating intermediates have been identified and tentatively mapped onto the CD chemical mechanism originally proposed by Dean, White, and co-workers [Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714-4720]. The first intermediate with lambda(max) approximately 350 nm is assigned as either a gem-diamine complex or a thiol adduct formed by nucleophilic attack of either the amine group or the sulfhydryl group of the substrate on the internal aldimine form of the pyridoxal 5'-phosphate (PLP) cofactor. The second intermediate, with absorption features at approximately 417 and approximately 340 nm, is assigned as Cys aldimine and Cys ketimine forms in rapid equilibrium. In agreement with this assignment, a significant substrate alpha-deuterium equilibrium isotope effect ((2)H-EIE) favoring the aldimine form (417 nm) is observed in the second state produced in either wild-type CD0387 or the inactive C326A variant protein, which lacks the nucleophilic cysteine residue and is thus unable to proceed beyond this state unless "rescued" by a high concentration of an exogenous thiol. The third intermediate has an additional approximately 506 nm feature, characteristic of a quinonoid form, along with the features of the previous state. Its assignment as Ala aldimine, quinonoid, and ketimine forms in rapid equilibrium, which associates its formation with C-S bond cleavage and persulfide formation, is supported by its failure to develop in the C326A variant and the normal kinetic isotope effect ((2)H-KIE) on its formation, which is similar in magnitude to the (2)H-EIE disfavoring Cys-ketimine (from which the third state forms) in the second state. Decay of the Ala quinonoid absorption is tentatively attributed to a conformational change by the enzyme that disfavors this form in its equilibrium with Ala aldimine and Ala ketimine. Subsequent decay of the ketimine absorption ( approximately 340 nm) is attributed to release of Ala from the cofactor with an observed rate constant of 10 s(-1), the slowest step in the persulfide-forming half-reaction. The enzyme-persulfide.Ala complex dissociates rapidly with a K(d) of 98 mM. The final state with lambda(max) approximately 350 nm is assigned as a dead-end complex between the enzyme-persulfide and a second l-cysteine, which adds to the cofactor via its sulfhydryl group, possibly forming a cyclic thiazolidine species.