The accessibility of the active site and conformation states of the beta 2 subunit of tryptophan synthase studied by fluorescence quenching

The rate of quenching of the fluorescence of pyridoxal 5'-phosphate in the active site of the beta 2 subunit of tryptophan synthase from Escherichia coli was measured to estimate the accessibility of the coenzyme to the small molecules iodide and acrylamide. The alpha subunit and the substrate L-serine substantially reduced the quenching rate. For iodide, the order of decreasing quenching was: Schiff's base of N alpha-acetyl-lysine with pyridoxal 5'-phosphate greater than holo beta 2 subunit greater than holo alpha 2 beta 2 complex approximately equal to holo beta 2 subunit + L-serine greater than holo alpha 2 beta 2 complex + L-serine. The coenzyme in the beta 2 subunit is apparently freely accessible to both iodide and acrylamide (kappa approximately equal to 2 X 10(9) M-1 s-1), but the alpha subunit and L-serine decrease the rate by factors of 2-5. Quenching of the fluorescence of the single tryptophan residue of the beta 2 subunit revealed that the apo and holo forms exist in different states, whereas the alpha subunit stabilizes a third conformation. As the alpha subunit binds to the beta 2 subunit, the tryptophan residue, which is within 2.2 nm of the active site of the beta 2 subunit, probably rotates with respect to the plane of the ring of the coenzyme, such that fluorescence energy transfer from tryptophan to pyridoxal phosphate is greatly reduced. The alpha subunit strongly protects the active-site ligand indole propanol phosphate from quenching with acrylamide, consistent with the active site being deep in a cleft in the protein. Iodide induces dissociation of the holo alpha 2 beta 2 complex [E. W. Miles & M. Moriguchi (1977) J. Biol. Chem. 252, 6594-6599]. The effect of iodide on the fluorescence properties of holo alpha 2 beta 2 complex allows us to estimate an upper limit for the dissociation constant for the alpha 2 beta 2 complex of 10(-8) M, in the absence of iodide.     

Energetic adaptation of ligand binding to subunit structure of tryptophan synthase from Escherichia coli

The binding of indole and L-serine to the isolated alpha and beta 2 subunits and the native alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli was investigated by direct microcalorimetry to reveal the energetic adaptation of ligand binding to the subunit structure of a multienzyme complex. In contrast to the general finding that negative heat capacity changes are associated with ligand binding to proteins, complex formation of indole and the alpha subunit involves a small positive change in heat capacity. This unusual result was considered as being indicative of a loosening of the protein structure. Such an interpretation is in good agreement with results of chemical accessibility studies (Freedberg & Hardman, 1971). Whereas the thermodynamic parameters of indole binding are not influenced by the subunit interaction, the large negative change in heat capacity of -6.5 kJ/(K X mol of beta 2) measured for the binding of L-serine to the isolated beta 2 subunit disappears completely when serine interacts with the tetrameric complex. These data demonstrate that the energy transduction pattern and therefore the functional roles of the substrates indole and L-serine vary strongly with the subunit structure of tryptophan synthase.     

Subunit interaction in tryptophan synthase of Escherichia coli: calorimetric studies on association of alpha and beta 2 subunits.

Association of the apo-beta 2 and the holo-(beta-PLP)2 subunits of tryptophan synthase from Escherichia coli (L-serine hydro-lyase (adding indole) (EC 4.2.1.20)) with alpha subunits of the same enzyme has been studied by microcalorimetry. The results obtained from thermometric titrations clearly demonstrate that only the native complex alpha2beta 2 is formed, independent of an excess of alpha protein. The reaction of the holo-(beta-PLP)2 with alpha subunits at 25 degrees C is accompanied by a negative enthalpy change, which is almost twice as large as that for complex formation with the apo-beta 2 protein, thus indicating that the interaction enthalpy becomes more favorable in the presence of the coenzyme pyridoxal 5'-phosphate (PLP). Both reaction enthalpies show very large negative temperature coefficients, -3600 +/- 100 cal K-1 (Mol of beta 2)-1 being the value for the formation of the apoenzyme and -2300 +/- 100 cal K-1 (mol of beta 2)-1 pertaining to formation of the holoenzyme. The studies on the association of alpha and beta2 subunits in the two buffers revealed that at 25 degrees C approximately 0.75 proton are absorbed in the presence and absence of the coenzyme, whereas at 35 degrees C one proton is taken up from the solution when PLP is present, but two if the apo-beta 2 complex reacts. These results are a clear indication of energetic linkage between intersubunit interaction, hydrogen ion equilibria, and the binding of the coenzyme.     

L-serine binds to arginine-148 of the beta 2 subunit of Escherichia coli tryptophan synthase

Inactivation of the beta 2 subunit and of the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli by the arginine-specific dicarbonyl reagent phenylglyoxal results from modification of one arginyl residue per beta monomer. The substrate L-serine protects the holo beta 2 subunit and the holo alpha 2 beta 2 complex from both inactivation and arginine modification but has no effect on the inactivation or modification of the apo forms of the enzyme. This result and the finding that phenylglyoxal competes with L-serine in reactions catalyzed by both the holo beta 2 subunit and the holo alpha 2 beta 2 complex indicate that L-serine and phenylglyoxal both bind to the same essential arginyl residue in the holo beta 2 subunit. The apo beta 2 subunit is protected from phenylglyoxal inactivation much more effectively by phosphopyridoxyl-L-serine than by either pyridoxal phosphate or pyridoxine phosphate, both of which lack the L-serine moiety. The phenylglyoxal-modified apo beta 2 subunit binds pyridoxal phosphate and the alpha subunit but cannot bind L-serine or L-tryptophan. We conclude that the alpha-carboxyl group of L-serine and not the phosphate of pyridoxal phosphate binds to the essential arginyl residue in the beta 2 subunit. The specific arginyl residue in the beta 2 subunit which is protected by L-serine from modification by phenyl[2-14C]glyoxal has been identified as arginine-148 by isolating a labeled cyanogen bromide fragment (residues 135-149) and by digesting this fragment with pepsin to yield the labeled dipeptide arginine-methionine (residues 148-149). The primary sequence near arginine-148 contains three other basic residues (lysine-137, arginine-141, and arginine-150) which may facilitate anion binding and increase the reactivity of arginine-148. The conservation of the arginine residues 141, 148, and 150 in the sequences of tryptophan synthase from E. coli, Salmonella typhimurium, and yeast supports a functional role for these three residues in anion binding. The location and role of the active-site arginyl residues in the beta 2 subunit and in two other enzymes which contain pyridoxal phosphate, aspartate aminotransferase and glycogen phosphorylase, are compared.     

Structure and mechanism of Escherichia coli pyridoxine 5'-phosphate oxidase

Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes the oxidation of either pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP), forming pyridoxal 5'-phosphate (PLP). This reaction serves as the terminal step in the de novo biosynthesis of PLP in E. coli and as a part of the salvage pathway of this coenzyme in both E. coli and mammalian cells. Recent studies have shown that in addition to the active site, PNPOx contains a noncatalytic site that binds PLP tightly. The crystal structures of PNPOx with one and two molecules of PLP bound have been determined. In the active site, the PLP pyridine ring is stacked almost parallel against the re-face of the middle ring of flavin mononucleotide (FMN). A large protein conformational change occurs upon binding of PLP. When the protein is soaked with excess PLP an additional molecule of this cofactor is bound about 11 A from the active site. A possible tunnel exists between the two sites. Site mutants were made of all residues at the active site that make interactions with the substrate. Stereospecificity studies showed that the enzyme is specific for removal of the proR hydrogen atom from the prochiral C4' carbon of PMP. The crystal structure and the stereospecificity studies suggest that the pair of electrons on C4' of the substrate are transferred to FMN as a hydride ion.     

Vitamin B(6) salvage enzymes: mechanism, structure and regulation

Vitamin B(6) is a generic term referring to pyridoxine, pyridoxamine, pyridoxal and their related phosphorylated forms. Pyridoxal 5'-phosphate is the catalytically active form of vitamin B(6), and acts as cofactor in more than 140 different enzyme reactions. In animals, pyridoxal 5'-phosphate is recycled from food and from degraded B(6)-enzymes in a "salvage pathway", which essentially involves two ubiquitous enzymes: an ATP-dependent pyridoxal kinase and an FMN-dependent pyridoxine 5'-phosphate oxidase. Once it is made, pyridoxal 5'-phosphate is targeted to the dozens of different apo-B(6) enzymes that are being synthesized in the cell. The mechanism and regulation of the salvage pathway and the mechanism of addition of pyridoxal 5'-phosphate to the apo-B(6)-enzymes are poorly understood and represent a very challenging research field. Pyridoxal kinase and pyridoxine 5'-phosphate oxidase play kinetic roles in regulating the level of pyridoxal 5'-phosphate formation. Deficiency of pyridoxal 5'-phosphate due to inborn defects of these enzymes seems to be involved in several neurological pathologies. In addition, inhibition of pyridoxal kinase activity by several pharmaceutical and natural compounds is known to lead to pyridoxal 5'-phosphate deficiency. Understanding the exact role of vitamin B(6) in these pathologies requires a better knowledge on the metabolism and homeostasis of the vitamin. This article summarizes the current knowledge on structural, kinetic and regulation features of the two enzymes involved in the PLP salvage pathway. We also discuss the proposal that newly formed PLP may be transferred from either enzyme to apo-B(6)-enzymes by direct channeling, an efficient, exclusive, and protected means of delivery of the highly reactive PLP. This new perspective may lead to novel and interesting findings, as well as serve as a model system for the study of macromolecular channeling. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.     

Influence of cofactor pyridoxal 5'-phosphate on reversible high-pressure denaturation of isolated beta 2 dimer of tryptophan synthase bienzyme complex from Escherichia coli

High hydrostatic pressure has been shown to cause reversible dissociation of the isolated apo beta 2 dimer of tryptophan synthase from Escherichia coli into enzymatically inactive monomers [Seifert, T., Bartholmes, P., & Jaenicke, R. (1982) Biophys. Chem. 15, 1-8]. Addition of the coenzyme pyridoxal 5'-phosphate affects the structural stability, as well as the kinetics of dissociation and deactivation. The apo beta 2 dimer is deactivated faster than the holoenzyme by a factor of 10. The midpoints of the corresponding equilibrium transition curves are observed at 690 and 870 bar, respectively. As shown by hybridization of native and chemically modified beta chains, the loss of enzymatic activity is accompanied by subunit dissociation. An additional deactivating effect is produced by the pressure-induced release of the cofactor from the holoenzyme. Renaturation after decompression has been monitored by circular dichroism and intrinsic fluorescence emission. Alterations of the dichroic absorption at 222 nm reflect the recovery of the native secondary structure, while tryptophan fluorescence represents a specific probe for the native tertiary structure in the immediate neighborhood of the active center of the enzyme. By application of both methods to monitor the reconstitution of the apo beta 2 dimer, two first-order processes may be separated along the time scale. The faster phase (k1 = 1.2 X 10(-2) s-1) yields a "structured monomer" with 85% native secondary structure and the tryptophan side chain buried in its native hydrophobic environment. As indicated by sodium borohydride reduction, this intermediate is able to interact with the coenzyme pyridoxal 5'-phosphate in the correct way; however, it does not show enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tight binding of pyridoxal 5'-phosphate to recombinant Escherichia coli pyridoxine 5'-phosphate oxidase

Escherichia coli pyridoxine (pyridoxamine) 5'-phosphate oxidase (PNPOx) catalyzes the oxidation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate to pyridoxal 5'-phosphate (PLP) using flavin mononucleotide (FMN) as the immediate electron acceptor and oxygen as the ultimate electron acceptor. This reaction serves as the terminal step in the de novo biosynthesis of PLP in E. coli. Removal of FMN from the holoenzyme results in a catalytically inactive apoenzyme. PLP molecules bind tightly to both apo- and holoPNPOx with a stoichiometry of one PLP per monomer. The unique spectral property of apoPNPOx-bound PLP suggests a non-Schiff base linkage. HoloPNPOx with tightly bound PLP shows normal catalytic activity, suggesting that the tightly bound PLP is at a noncatalytic site. The tightly bound PLP is readily transferred to aposerine hydroxymethyltransferase in dilute phosphate buffer. However, when the PNPOx. PLP complex was added to aposerine hydroxymethyltransferase suspended in an E. coli extract the rate of reactivation of the apoenzyme was several-fold faster than when free PLP was added. This suggests that PNPOx somehow targets PLP to aposerine hydroxymethyltransferase in vivo.     

Structure and function of the tryptophan synthase alpha(2)beta(2) complex. Roles of beta subunit histidine 86

To probe the structural and functional roles of active-site residues in the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium, we have determined the effects of mutation of His(86) in the beta subunit. His(86) is located adjacent to beta subunit Lys(87), which forms an internal aldimine with the pyridoxal phosphate and catalyzes the abstraction of the alpha-proton of L-serine. The replacement of His(86) by leucine (H86L) weakened pyridoxal phosphate binding approximately 20-fold and abolished the circular dichroism signals of the bound coenzyme and of a reaction intermediate. Correlation of these results with previous crystal structures indicates that beta-His(86) plays a structural role in binding pyridoxal phosphate and in stabilizing the correct orientation of pyridoxal phosphate in the active site of the beta subunit. The H86L mutation also altered the pH profiles of absorbance and fluorescence signals and shifted the pH optimum for the synthesis of L-tryptophan from pH 7.5 to 8.8. We propose that the interaction of His(86) with the phosphate of pyridoxal phosphate and with Lys(87) lowers the pK(a) of Lys(87) in the wild-type alpha(2)beta(2) complex and thereby facilitates catalysis by Lys(87) in the physiological pH range.     

Three-dimensional structure of the tryptophan synthase alpha 2 beta 2 multienzyme complex from Salmonella typhimurium

The three-dimensional structure of the alpha 2 beta 2 complex of tryptophan synthase from Salmonella typhimurium has been determined by x-ray crystallography at 2.5 A resolution. The four polypeptide chains are arranged nearly linearly in an alpha beta beta alpha order forming a complex 150 A long. The overall polypeptide fold of the smaller alpha subunit, which cleaves indole glycerol phosphate, is that of an 8-fold alpha/beta barrel. The alpha subunit active site has been located by difference Fourier analysis of the binding of indole propanol phosphate, a competitive inhibitor of the alpha subunit and a close structural analog of the natural substrate. The larger pyridoxal phosphate-dependent beta subunit contains two domains of nearly equal size, folded into similar helix/sheet/helix structures. The binding site for the coenzyme pyridoxal phosphate lies deep within the interface between the two beta subunit domains. The active sites of neighboring alpha and beta subunits are separated by a distance of about 25 A. A tunnel with a diameter matching that of the intermediate substrate indole connects these active sites. The tunnel is believed to facilitate the diffusion of indole from its point of production in the alpha subunit active site to the site of tryptophan synthesis in the beta active site and thereby prevent its escape to the solvent during catalysis.     

Active-site modification of mammalian pyruvate dehydrogenase by pyridoxal 5'-phosphate

The pyruvate dehydrogenase multienzyme complex from bovine kidney and heart is inactivated by treatment with pyridoxal 5'-phosphate and sodium cyanide or sodium borohydride. The site of this inhibition is the pyruvate dehydrogenase (E1) component of the complex. Inactivation of E1 by the pyridoxal phosphate-cyanide treatment was prevented by thiamin pyrophosphate. Equilibrium binding studies showed that E1 contains two thiamin pyrophosphate binding sites per molecule (alpha 2 beta 2) and that modification of E1 increased the dissociation constant (Kd) for thiamin pyrophosphate about 5-fold. Incorporation of approximately 2.4 equiv of 14CN per mole of E1 tetramer in the presence of pyridoxal phosphate resulted in about a 90% loss of E1 activity. Radioactivity was incorporated predominantly into the E1 alpha subunit. Radioactive N6-pyridoxyllysine was identified in an acid hydrolysate of the E1-pyridoxal phosphate complex that had been reduced with NaB3H4. The data are interpreted to indicate that in the presence of sodium cyanide or sodium borohydride, pyridoxal phosphate reacts with a lysine residue at or near the thiamin pyrophosphate binding site of E1. This binding site is apparently located on the alpha subunit.     

X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with FMN at 1.8 A resolution

BACKGROUND: Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate (PLP), a cofactor used by many enzymes involved in amino acid metabolism. The enzyme oxidizes either the 4'-hydroxyl group of pyridoxine 5'-phosphate (PNP) or the 4'-primary amine of pyridoxamine 5'-phosphate (PMP) to an aldehyde. PNPOx is a homodimeric enzyme with one flavin mononucleotide (FMN) molecule non-covalently bound to each subunit. A high degree of sequence homology among the 15 known members of the PNPOx family suggests that all members of this group have similar three-dimensional folds. RESULTS: The crystal structure of PNPOx from E. coli has been determined to 1.8 A resolution. The monomeric subunit folds into an eight-stranded beta sheet surrounded by five alpha-helical structures. Two monomers related by a twofold axis interact extensively along one-half of each monomer to form the dimer. There are two clefts at the dimer interface that are symmetry-related and extend from the top to the bottom of the dimer. An FMN cofactor that makes interactions with both subunits is located in each of these two clefts. CONCLUSIONS: The structure is quite similar to the recently deposited 2.7 A structure of Saccharomyces cerevisiae PNPOx and also, remarkably, shares a common structural fold with the FMN-binding protein from Desulfovibrio vulgaris and a domain of chymotrypsin. This high-resolution E. coli PNPOx structure permits predictions to be made about residues involved in substrate binding and catalysis. These predictions provide testable hypotheses, which can be answered by making site-directed mutants.     

The molecular pathway for the allosteric regulation of tryptophan synthase

The pyridoxal 5'-phosphate (PLP)-dependent tryptophan synthase is a alpha(2)beta(2) complex. The alpha-beta subunit interaction plays a critical role both in the reciprocal activation of the individual subunits and in the allosteric regulation. We have investigated whether mutations of alpha loop6 Gly(181) and beta helix6 Ser(178) affect intersubunit communication. The loss of the hydrogen bond between these residues, achieved by proline substitution, does not significantly influence the intersubunit catalytic activation, but completely abolishes ligand-induced intersubunit signaling. The comparison of the crystal structure of the wild type and beta Ser(178)Pro mutant, in the absence and presence of alpha-subunit ligands, indicates that the removal of the interaction between beta Ser(178) and alpha Gly(181) strongly affects the equilibrium between active (closed) and inactive (open) conformations of the alpha-active site, the latter being stabilized in both mutants.     

Structure of P-protein of the glycine cleavage system: implications for nonketotic hyperglycinemia

The crystal structure of the P-protein of the glycine cleavage system from Thermus thermophilus HB8 has been determined. This is the first reported crystal structure of a P-protein, and it reveals that P-proteins do not involve the alpha(2)-type active dimer universally observed in the evolutionarily related pyridoxal 5'-phosphate (PLP)-dependent enzymes. Instead, novel alphabeta-type dimers associate to form an alpha(2)beta(2) tetramer, where the alpha- and beta-subunits are structurally similar and appear to have arisen by gene duplication and subsequent divergence with a loss of one active site. The binding of PLP to the apoenzyme induces large open-closed conformational changes, with residues moving up to 13.5 A. The structure of the complex formed by the holoenzyme bound to an inhibitor, (aminooxy)acetate, suggests residues that may be responsible for substrate recognition. The molecular surface around the lipoamide-binding channel shows conservation of positively charged residues, which are possibly involved in complex formation with the H-protein. These results provide insights into the molecular basis of nonketotic hyperglycinemia.     

Rv2607 from Mycobacterium tuberculosis is a pyridoxine 5'-phosphate oxidase with unusual substrate specificity

Despite intensive effort, the majority of the annotated Mycobacterium tuberculosis genome consists of genes encoding proteins of unknown or poorly understood function. For example, there are seven conserved hypothetical proteins annotated as homologs of pyridoxine 5'-phosphate oxidase (PNPOx), an enzyme that oxidizes pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP) to form pyridoxal 5'-phosphate (PLP). We have characterized the function of Rv2607 from Mycobacterium tuberculosis H37Rv and shown that it encodes a PNPOx that oxidizes PNP to PLP. The k(cat) and K(M) for this reaction were 0.01 s(-1) and 360 μM, respectively. Unlike many PNPOx enzymes, Rv2607 does not recognize PMP as a substrate.     

Conformational effects of ligand binding on the beta 2 subunit of Escherichia coli tryptophan synthase analyzed with monoclonal antibodies

Five monoclonal antibodies recognizing five different epitopes of the native beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.1.2.20) were used to analyze the conformational changes occurring upon ligand binding or chemical modifications of the enzyme. For this purpose, the affinities of each antibody for the different forms of the enzyme were determined by using an enzyme-linked immunosorbent assay which allows measurement of the dissociation constant of antigen-antibody equilibrium in solution. The fixation of the coenzyme pyridoxal 5'-phosphate and the substrate L-serine modifies the affinity constants of most of the antibodies for the enzyme, thus showing the existence of extended conformational rearrangements of the protein. The association of the alpha subunit with the beta 2 subunit, which brings about an increase of the tryptophan synthase activity and abolishes the serine deaminase activity of beta 2, is accompanied by an important conformational change of the N-terminal domain of beta 2 (F1) since none of the anti-F1 monoclonal antibodies can bind to alpha 2 beta 2. Similarly, chemical modifications of beta 2 which are known to produce significant effects on the enzymatic activities of beta 2 result in changes of the affinities of the monoclonal antibodies which can be interpreted as the acquisition of different conformational states of the enzyme.     

Kinetics of the spontaneous transient unfolding of a native protein studied with monoclonal antibodies. Monomer/dimer transition in the tryptophan-synthase beta 2 subunit

Included in a series of monoclonal antibodies obtained after immunization with the native holo beta 2 subunit of tryptophan synthase of Escherichia coli (EC 4.2.1.20), are some that interact preferentially with a denatured state of the antigen (Friguet et al., 1984). A study of the equilibrium and kinetic characteristics of the interaction of one of these antibodies with native apo beta 2 (i.e. free of pyridoxal 5'-phosphate) and with one of its proteolytic domains is reported here. The antibody is shown to interact strongly with the isolated domain in accordance with a simple equilibrium. In the presence of native beta 2, the antibody binds exclusively to the dissociated beta-monomer. The interaction of this antibody with native apo beta 2 is used to determine the equilibrium and kinetic constants of the monomer-dimer equilibrium. The values obtained are 4.5 X 10(-8) M for the equilibrium constant and 7.9 X 10(-3) s-1 for the rate constant of the dissociation of apo beta 2 into beta-monomers.     

Studies on the changes in protein fluorescence and enzymic activity of aspartate aminotransferase on binding of pyridoxal 5′-phosphate

1. The alpha and beta subforms of aspartate aminotransferase were purified from pig heart. 2. The alpha subform contained 2mol of pyridoxal 5'-phosphate. The apo-(alpha subform) could be fully reactived by combination with 2mol of cofactor. 3. The protein fluorescence of the apo-(alpha subform) decreased non-linearly with increase in enzyme activity and concentration of bound cofactor. 4. It is concluded that the enzyme activity/mol of bound cofactor is largely independent of the number of cofactors bound to the dimer. 5. The beta subform had approximately half the specific enzyme activity of the alpha subform, and contained an average of one active pyridoxal 5'-phosphate molecule per molecule, which could be removed by glutamate, and another inactive cofactor which could only be removed with NaOH. 6. On recombination with pyridoxal 5'-phosphate the protein fluorescence of the apo-(beta subform) decreased linearly, showing that each dimeric enzyme molecule contained one active and one inactive bound cofactor. 7. The results are not consistent with a flip-flop mechanism for this enzyme.     

Mechanism for multiple-substrates recognition of alpha-aminoadipate aminotransferase from Thermus thermophilus

Alpha-aminoadipate aminotransferase (AAA-AT), a homolog of mammalian kynurenine aminotransferase II (Kat II), transfers an amino group to 2-oxoadipate to yield alpha-aminoadipate in lysine biosynthesis through the alpha-aminoadipate pathway in Thermus thermophilus. AAA-AT catalyzes transamination against various substrates, including AAA, glutamate, leucine, and aromatic amino acids. To elucidate the structural change for recognition of various substrates, we determined crystal structures of AAA-AT in four forms: with pyridoxal 5'-phosphate (PLP) (PLP complex), with PLP and leucine (PLP/Leu complex), with N-phosphopyridoxyl-leucine (PPL) (PPL complex), and with N-phosphopyridoxyl-alpha-aminoadipate (PPA) at 2.67, 2.26, 1.75, and 1.67 A resolution, respectively. The PLP complex is in an open state, whereas PLP/Leu, PPL, and PPA complexes are in closed states with maximal displacement (over 7 A) of the alpha2 helix and the beta1 strand in the small domain to cover the active site, indicating that conformational change is induced by substrate binding. In PPL and PLP/Leu complexes, several hydrophobic residues on the alpha2 helix recognize the hydrophobic side chain of the bound leucine moiety whereas, in the PPA complex, the alpha2 helix rotates to place the guanidium moiety of Arg23 on the helix at the appropriate position to interact with the carboxyl side chain of the AAA moiety. These results indicate that AAA-AT can recognize various kinds of substrates using the mobile alpha2 helix. The crystal structures and site-directed mutagenesis revealed that intersubunit-electrostatic interactions contribute to the elevated thermostability of this enzyme.     

Physical and kinetic properties of a pyridoxal reductase purified from bakers' yeast

Pyridoxine dehydrogenase (1.1.1.65) (pyridoxal reductase), purified to homogeneity from baker's yeast, is a monomer of Mr approximately 33,000. It catalyzes the reversible oxidation of pyridoxine by NADP to yield pyridoxal and NADPH; equilibrium lies far in the direction of pyridoxine formation (Keq approximately 1.4 X 10(11) l/mol at 25 degrees C). Reduction of pyridoxal occurs most rapidly at pH 6.0-7.0; oxidation of pyridoxine is optimal at pH 8.6. NAD and NADH do not replace NADP and NADPH as substrates; pyridoxine, pyridoxal and pyridoxal 5'-phosphate are the only naturally occurring cosubstrates found. Several other aromatic aldehydes also are reduced, but substrate specificity and other properties of the enzyme distinguish it clearly from other alcohol dehydrogenases or aldehyde reductases. Between pH 6.3 and 7.1 (the intracellular pH of yeast), V/Km with pyridoxal and NADPH as substrates is greater than 600 times that observed with pyridoxine and NADPH as substrates is greater than 600 times that observed with pyridoxine and NADP as substrates. These and other considerations strongly indicate that the dehydrogenase functions in vivo to reduce pyridoxal to pyridoxine, which is the preferred substrate for pyridoxal (pyridoxine) kinase in yeast.