Tyrosine 70 increases the coenzyme affinity of aspartate aminotransferase. A site-directed mutagenesis study

The crucial step in enzymatic transamination is the tautomerization of aldimine/ketimine intermediates, formed between the pyridoxyl coenzyme and the amino/keto acid substrate, which is catalyzed primarily by the active site residue Lys-258 (Malcolm, B. A., and Kirsch, J. F. (1985) Biochem. Biophys. Res. Commun. 132, 915-921; W. L. Finlayson and J. F. Kirsch, in preparation). Tyr-70 is localized in close proximity to Lys-258 and, in addition, forms a hydrogen bond with the coenzyme phosphate. Tyr-70 has been postulated to have an important role in the tautomerization (Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H., and Christen, P. (1984) J. Mol. Biol. 174, 497-525). This hypothesis has now been tested by the construction and analysis of a mutant Escherichia coli aspartate aminotransferase in which Tyr-70 has been changed to Phe (Y70F). Y70F retains at least 15% of the maximal activity of the wild type enzyme (WT) (kcat = 170 +/- 15 s-1 for WT versus greater than or equal to 26 +/- 3 s-1 for Y70F and shows increased Michaelis constants for both substrates (KmAsp = 2.5 +/- 0.4 mM; Km alpha Kg = 0.59 +/- 0.08 mM for WT versus KmAsp = 3.9 +/- 0.3 mM; Km alpha Kg = 2.70 +/- 0.02 mM for Y70F (where alpha Kg is alpha-ketoglutarate) ). The spectrophotometrically determined pK a values of the internal aldimines formed between pyridoxal 5'-phosphate (PLP) and Lys-258 are identical for WT and Y70F. In assays where excess L-aspartate and excess PLP are incubated with either WT or Y70F, the mutant enzyme converts the free PLP to free pyridoxamine 5'-phosphate 80-fold faster than WT (k = (3.75 +/- 0.23) X 10(-2)s-1 for Y70F versus (4.90 +/- 0.02) X 10(-4)s-1 for WT). Y70F also converts free pyridoxamine 5'-phosphate to free PLP faster than WT. Thus, Y70F dissociates coenzyme more readily than does WT. It therefore appears that the role of Tyr-70 is mainly in preventing the dissociation of the coenzyme from the enzyme. Tyr-70 does not function in an essential chemical step.     

Crystal structure of histidinol phosphate aminotransferase (HisC) from Escherichia coli, and its covalent complex with pyridoxal-5'-phosphate and l-histidinol phosphate

The biosynthesis of histidine is a central metabolic process in organisms ranging from bacteria to yeast and plants. The seventh step in the synthesis of histidine within eubacteria is carried out by a pyridoxal-5'-phosphate (PLP)-dependent l-histidinol phosphate aminotransferase (HisC, EC 2.6.1.9). Here, we report the crystal structure of l-histidinol phosphate aminotransferase from Escherichia coli, as a complex with pyridoxamine-5'-phosphate (PMP) at 1.5 A resolution, as the internal aldimine with PLP, and in a covalent, tetrahedral complex consisting of PLP and l-histidinol phosphate attached to Lys214, both at 2.2 A resolution. This covalent complex resembles, in structural terms, the gem-diamine intermediate that is formed transiently during conversion of the internal to external aldimine.HisC is a dimeric enzyme with a mass of approximately 80 kDa. Like most PLP-dependent enzymes, each HisC monomer consists of two domains, a larger PLP-binding domain having an alpha/beta/alpha topology, and a smaller domain. An N-terminal arm contributes to the dimerization of the two monomers. The PLP-binding domain of HisC shows weak sequence similarity, but significant structural similarity with the PLP-binding domains of a number of PLP-dependent enzymes. Residues that interact with the PLP cofactor, including Tyr55, Asn157, Asp184, Tyr187, Ser213, Lys214 and Arg222, are conserved in the family of aspartate, tyrosine and histidinol phosphate aminotransferases. The imidazole ring of l-histidinol phosphate is bound, in part, through a hydrogen bond with Tyr110, a residue that is substituted by Phe in the broad substrate specific HisC enzymes from Zymomonas mobilis and Bacillus subtilis.Comparison of the structures of the HisC internal aldimine, the PMP complex and the HisC l-histidinol phosphate complex reveal minimal changes in protein or ligand structure. Proton transfer, required for conversion of the gem-diamine to the external aldimine, does not appear to be limited by the distance between substrate and lysine amino groups. We propose that the tetrahedral complex has resulted from non-productive binding of l-histidinol phosphate soaked into the HisC crystals, resulting in its inability to be converted to the external aldimine at the HisC active site.     

NMR studies of the stability, protonation States, and tautomerism of (13)C- AND (15)N-labeled aldimines of the coenzyme pyridoxal 5'-phosphate in water

We have measured the pH-dependent (1)H, (13)C, and (15)N NMR spectra of pyridoxal 5'-phosphate ((13)C(2)-PLP) mixed with equal amounts of either doubly (15)N-labeled diaminopropane, (15)N(α)-labeled l-lysine, or (15)N(ε)-labeled l-lysine as model systems for various intermediates of the transimination reaction in PLP-dependent enzymes. At low pH, only the hydrate and aldehyde forms of PLP and the free protonated diamines are present. Above pH 4, the formation of single- and double-headed aldimines (Schiff bases) with the added diamines is observed, and their (13)C and (15)N NMR parameters have been characterized. For 1:1 mixtures the single-headed aldimines dominate. In a similar way, the NMR parameters of the geminal diamine formed with diaminopropane at high pH are measured. However, no geminal diamine is formed with l-lysine. In contrast to the aldimine formed with the ε-amino group of lysine, the aldimine formed with the α-amino group is unstable at moderately high pH but dominates slightly below pH 10. By analyzing the NMR data, both the mole fractions of the different PLP species and up to 6 different protonation states including their pK(a) values were obtained. Furthermore, the data show that all Schiff bases are subject to a proton tautomerism along the intramolecular OHN hydrogen bond, where the zwitterionic form is favored before deprotonation occurs at high pH. This observation, as well as the observation that around pH 7 the different PLP species are present in comparable amounts, sheds new light on the mechanism of the transimination reaction.     

Crystal structure at 1.45 A resolution of alanine racemase from a pathogenic bacterium, Pseudomonas aeruginosa, contains both internal and external aldimine forms

The structure of the catabolic alanine racemase, DadX, from the pathogenic bacterium Pseudomonas aeruginosa, reported here at 1.45 A resolution, is a dimer in which each monomer is comprised of two domains, an eight-stranded alpha/beta barrel containing the PLP cofactor and a second domain primarily composed of beta-strands. The geometry of each domain is very similar to that of Bacillus stearothermophilus alanine racemase, but the rotation between domains differs by about 15 degrees. This change does not alter the structure of the active site in which almost all residues superimpose well with a low rms difference of 0.86 A. Unexpectedly, the active site of DadX contains a guest substrate that is located where acetate and propionate have been observed in the Bacillus structures. It is modeled as d-lysine and oriented such that its terminal NZ atom makes a covalent bond with C4' of PLP. Since the internal aldimine bond between the protein lysine, Lys33, and C4' of PLP is also unambiguously observed, there appears to be an equilibrium between both internally and externally reacted forms. The PLP cofactor adopts two partially occupied conformational states that resemble previously reported internal and external aldimine complexes.     

Substitution of an arginyl residue for the active site lysyl residue (Lys258) of aspartate aminotransferase

The active site lysyl residue (Lys258) of E. coli aspartate amino transferase was substituted for an arginyl residue by oligonucleotide-directed, site-specific mutagenesis. The mutant enzyme was obviously unable to form an aldimine bond with pyridoxal 5'-phosphate but firmly bound the coenzyme. The finding that the mutation did not lead to entire loss in the enzymic activity suggests that Lys258 may not be essential but auxiliary for enzymic catalysis. It is also conceived that the positive charge provided by Arg258 may contribute to the enzymic catalysis.     

The stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate is abolished in (Lys258----Ala) aspartate aminotransferase

Reconstitution of wild-type apoaspartate aminotransferase from Escherichia coli with [4'-3H]pyridoxamine 5'-phosphate results in stereospecific release of the pro-S C-4' 3H to the solvent. The reaction follows first-order kinetics (t1/2 = 15 min at pH 7.5 and 25 degrees C), its rate constant being similar to that found previously with mitochondrial aspartate aminotransferase from chicken (Tobler, H.P., Christen, P., and Gehring, H. (1986) J. Biol. Chem. 261, 7105-7108). Substituting the active site residue Lys258 by alanine via site-directed mutagenesis yields a catalytically inactive enzyme (Malcolm, B. A., and Kirsch, J. F. (1985) Biochem. Biophys. Res. Commun. 132, 915-921). This mutant enzyme fails to release any measurable 3H from bound [4'-3H]pyridoxamine 5'-phosphate. The data are consistent with earlier proposals that Lys258 is indispensable for the ketimine/aldimine tautomerization, and corroborate the previous conclusion that 3H exchange from enzyme-bound pyridoxamine 5'-phosphate mechanistically corresponds to the deprotonation at C-4' of the ketimine intermediate during the transamination reaction.     

Histidine 282 in 5-aminolevulinate synthase affects substrate binding and catalysis

5-Aminolevulinate synthase (ALAS), the first enzyme of the heme biosynthetic pathway in mammalian cells, is a member of the alpha-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. In all structures of the enzymes of the -oxoamine synthase family, a conserved histidine hydrogen bonds with the phenolic oxygen of the PLP cofactor and may be significant for substrate binding, PLP positioning, and maintenance of the pKa of the imine nitrogen. In ALAS, replacing the equivalent histidine, H282, with alanine reduces the catalytic efficiency for glycine 450-fold and decreases the slow phase rate for glycine binding by 85%. The distribution of the absorbing 420 and 330 nm species was altered with an A420/A330 ratio increased from 0.45 to 1.05. This shift in species distribution was mirrored in the cofactor fluorescence and 300-500 nm circular dichroic spectra and likely reflects variation in the tautomer distribution of the holoenzyme. The 300-500 nm circular dichroism spectra of ALAS and H282A diverged in the presence of either glycine or aminolevulinate, indicating that the reorientation of the PLP cofactor upon external aldimine formation is impeded in H282A. Alterations were also observed in the K(Gly)d value and spectroscopic and kinetic properties, while the K(PLP)d increased 9-fold. Altogether, the results imply that H282 coordinates the movement of the pyridine ring with the reorganization of the active site hydrogen bond network and acts as a hydrogen bond donor to the phenolic oxygen to maintain the protonated Schiff base and enhance the electron sink function of the PLP cofactor.     

Identification of coenzyme aldimine proton in 1H NMR spectra of pyridoxal 5'-phosphate dependent enzymes: aspartate aminotransferase isoenzymes

The pyridoxal form of the alpha subform of cytosolic aspartate aminotransferase (EC 2.6.1.1) is fully active and binds pyridoxal 5'-phosphate via an aldimine formation with Lys-258 whereas the gamma subform is virtually inactive and lacks the aldimine linkage. Comparison of 1H NMR spectra between the alpha and gamma subforms suggested that peak 1 of the alpha subform at 8.89 ppm contains a resonance assignable to the internal aldimine 4'-H. Reaction with a reagent that cleaves or modifies the internal aldimine bond [(amino-oxy)acetate, L-cysteinesulfinate, NH2OH, NaBH4, or NaCNBH3] caused the disappearance of a resonance line at 8.89 ppm that possessed a broad line width and corresponded in intensity to a single proton. These reagents were also used successfully for the identification of the aldimine 4'-H resonance in the mitochondrial isoenzyme. In contrast to the cytosolic isoenzyme whose resonance for the 4'-H did not show any detectable change in chemical shift with pH, the corresponding resonance in the mitochondrial isoenzyme exhibited pH-dependent chemical shift change (8.84 ppm at pH 5 and 8.67 ppm at pH 8) with a pK value of 6.3, reflecting the interisozymic difference in the microenvironment provided for the internal aldimine. Validity of the signal assignment was further shown by the two findings: the resonance assigned to the 4'-H emerged upon conversion of the pyridoxamine into the pyridoxal form, and the resonance appeared upon reconstitution of the apoenzyme with [4'-1H]pyridoxal phosphate but not with [4'-2H]pyridoxal phosphate.     

Controlling reaction specificity in pyridoxal phosphate enzymes

Pyridoxal 5'-phosphate enzymes are ubiquitous in the nitrogen metabolism of all organisms. They catalyze a wide variety of reactions including racemization, transamination, decarboxylation, elimination, retro-aldol cleavage, Claisen condensation, and others on substrates containing an amino group, most commonly α-amino acids. The wide variety of reactions catalyzed by PLP enzymes is enabled by the ability of the covalent aldimine intermediate formed between substrate and PLP to stabilize carbanionic intermediates at Cα of the substrate. This review attempts to summarize the mechanisms by which reaction specificity can be achieved in PLP enzymes by focusing on three aspects of these reactions: stereoelectronic effects, protonation state of the external aldimine intermediate, and interaction of the carbanionic intermediate with the protein side chains present in the active site. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.     

Crystal structure of 3-amino-5-hydroxybenzoic acid (AHBA) synthase.

The biosynthesis of ansamycin antibiotics, including rifamycin B, involves the synthesis of an aromatic precursor, 3-amino-5-hydroxybenzoic acid (AHBA), which serves as starter for the assembly of the antibiotics' polyketide backbone. The terminal enzyme of AHBA formation, AHBA synthase, is a dimeric, pyridoxal 5'-phosphate (PLP) dependent enzyme with pronounced sequence homology to a number of PLP enzymes involved in the biosynthesis of antibiotic sugar moieties. The structure of AHBA synthase from Amycolatopsis mediterranei has been determined to 2.0 A resolution, with bound cofactor, PLP, and in a complex with PLP and an inhibitor (gabaculine). The overall fold of AHBA synthase is similar to that of the aspartate aminotransferase family of PLP-dependent enzymes, with a large domain containing a seven-stranded beta-sheet surrounded by alpha-helices and a smaller domain consisting of a four-stranded antiparallel beta-sheet and four alpha-helices. The uninhibited form of the enzyme shows the cofactor covalently linked to Lys188 in an internal aldimine linkage. On binding the inhibitor, gabaculine, the internal aldimine linkage is broken, and a covalent bond is observed between the cofactor and inhibitor. The active site is composed of residues from two subunits of AHBA synthase, indicating that AHBA synthase is active as a dimer.     

Acceleration of the substrate Calpha deprotonation by an analogue of the second substrate palmitoyl-CoA in Serine Palmitoyltransferase

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyzes the pyridoxal 5'-phosphate (PLP)-dependent decarboxylative condensation reaction of l-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The binding of l-serine alone to SPT leads to the formation of the external aldimine but does not produce a detectable amount of the quinonoid intermediate. However, the further addition of S-(2-oxoheptadecyl)-CoA, a nonreactive analogue of palmitoyl-CoA, caused the apparent accumulation of the quinonoid. NMR studies showed that the hydrogen-deuterium exchange at Calpha of l-serine is very slow in the SPT-l-serine external aldimine complex, but the rate is 100-fold increased by the addition of S-(2-oxoheptadecyl)-CoA, showing a remarkable substrate synergism in SPT. In addition, the observation that the nonreactive palmitoyl-CoA facilitated alpha-deprotonation indicates that the alpha-deprotonation takes place before the Claisen-type C-C bond formation, which is consistent with the accepted mechanism of the alpha-oxamine synthase subfamily enzymes. Structural modeling of both the SPT-l-serine external aldimine complex and SPT-l-serine-palmitoyl-CoA ternary complex suggests a mechanism in which the binding of palmitoyl-CoA to SPT induced a conformation change in the PLP-l-serine external aldimine so that the Calpha-H bond of l-serine becomes perpendicular to the plane of the PLP-pyridine ring and is favorable for the alpha-deprotonation. The model also proposed that the two alternative hydrogen bonding interactions of His(159) with l-serine and palmitoyl-CoA play an important role in the conformational change of the external aldimine. This is the unique mechanism of SPT that prevents the formation of the reactive intermediate before the binding of the second substrate.     

Modulation of the internal aldimine pK(a)'s of 1-aminocyclopropane-1-carboxylate synthase and aspartate aminotransferase by specific active site residues

The active sites of the homologous pyridoxal phosphate- (PLP-) dependent enzymes 1-aminocyclopropane-1-carboxylate (ACC) synthase and aspartate aminotransferase (AATase) are almost entirely conserved, yet the pK(a)'s of the two internal aldimines are 9.3 and 7.0, respectively, to complement the substrate pK(a)'s (S-adenosylmethionine pK(a) = 7.8 and aspartate pK(a) = 9.9). This complementation is required for maximum enzymatic activity in the physiological pH range. The most prominent structural difference in the active site is that Ile232 of ACC synthase is replaced by alanine in AATase. The I232A mutation was introduced into ACC synthase with a resulting 1.1 unit decrease (from 9.3 to 8.2) in the aldimine pK(a), thus identifying Ile232 as a major determinant of the high pK(a) of ACC synthase. The mutation also resulted in reduced k(cat) (0.5 vs 11 s(-1)) and k(cat)/K(m) values (5.0 x 10(4) vs 1.2 x 10(6) M(-1) s(-1)). The effect of the mutation is interpreted as the result of shortening of the Tyr233-PLP hydrogen bond. Addition of the Y233F mutation to the I232A ACC synthase to generate the double mutant I232A/Y233F raised the pK(a) from 8.2 to 8.8, because the Y233F mutation eliminates the hydrogen bond between that residue and PLP. The introduction of the retro mutation A224I into AATase raised the aldimine pK(a) of that enzyme from 6.96 to 7.16 and resulted in a decrease in single-turnover k(max) (108 vs 900 s(-1) for aspartate) and k(max)/K(m)(app) (7.5 x 10(4) vs 3.8 x 10(5) M(-1) s(-1)) values. The distance from the pyridine nitrogen of the cofactor to a conserved aspartate residue is 2.6 A in AATase and 3.8 A in ACC synthase. The D230E mutation introduced into ACC synthase to close this distance increases the aldimine pK(a) from 9.3 to 10.0, as would be predicted from a shortened hydrogen bond.     

Aminolevulinate synthase: lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in animals and some bacteria. Lysine-313 of the mouse erythroid aminolevulinate synthase was recently identified to be linked covalently to the pyridoxal 5'-phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2:1959-1965). Here we report on the effect of replacement of aminolevulinate synthase lysine-313 by alanine, histidine, and glycine, using site-directed mutagenesis. Mutant enzymes were purified to homogeneity, and the purification yields were similar to those of the wild-type enzyme. Although their absorption spectra indicate that the mutant enzymes bind pyridoxal 5'-phosphate, they bind noncovalently. However, addition of glycine to the mutant enzymes led to the formation of external aldimines. The formation of an external aldimine between the pyridoxal 5'-phosphate cofactor and the glycine substrate is the first step in the mechanism of the aminolevulinate synthase-catalyzed reaction. In contrast, lysine-313 is an essential catalytic residue, because the K313-directed mutant enzymes have no measurable activity. In summary, site-directed mutagenesis of the aminolevulinate synthase active-site lysine-313, to alanine (K313A), histidine (K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5'-phosphate cofactor and the glycine substrate to produce external aldimines, but which are inactive. This suggests that lysine-313 has a functional role in catalysis.     

Ligand binding induces a large conformational change in O-acetylserine sulfhydrylase from Salmonella typhimurium.

Covalent binding of L-methionine as an external aldimine to the pyridoxal 5'-phosphate-cofactor in the K41A mutant of O-acetylserine sulfhydrylase from Salmonella typhimurium induces a large conformational change in the protein. Methionine mimics the action of the substrate O-acetyl-L-serine during catalysis. The alpha-carboxylate moiety of L-methionine in external aldimine linkage with the active site pyridoxal 5'-phosphate forms a hydrogen bonding network to the "asparagine-loop" P67-T68-N69-G70 which adopts a different conformation than in the native protein. The side-chain nitrogen of Asn69 moves more than 7 A to make a hydrogen bond to the alpha-carboxylate group of the inhibitor. As the external aldimine is formed, the PLP tilts by 13 degrees along its longitudinal axis such that C4' moves toward the entrance to the active site and the side-chain of the methionine is directed toward the active site entrance. The local rearrangement acts as a trigger to induce a large global conformational change in the protein. A subdomain comprised of beta-strand 4, alpha-helix 3, beta-strand 5 and alpha-helix 4 moves towards the active site by a rotation of 7 degrees. This subdomain movement results in a reduction of the severe twist of its central beta-sheet and reduces the active site entrance to a small hole, giving access only to small molecules like sulfide, the second substrate, or acetate, the first product.     

High resolution crystal structures of human kynurenine aminotransferase‐I bound to PLP cofactor,and in complex with aminooxyacetate

In this study, we report two high‐resolution structures of the pyridoxal 5′ phosphate (PLP)‐dependent enzyme kynurenine aminotransferase‐I (KAT‐I). One is the native structure with the cofactor in the PLP form bound to Lys247 with the highest resolution yet available for KAT‐I at 1.28 Å resolution, and the other with the general PLP‐dependent aminotransferase inhibitor, aminooxyacetate (AOAA) covalently bound to the cofactor at 1.54 Å. Only small conformational differences are observed in the vicinity of the aldimine (oxime) linkage with which the PLP forms the Schiff base with Lys247 in the 1.28 Å resolution native structure, in comparison to other native PLP‐bound structures. We also report the inhibition of KAT‐1 by AOAA and aminooxy‐phenylpropionic acid (AOPP), with IC50s of 13.1 and 5.7 μM, respectively. The crystal structure of the enzyme in complex with the inhibitor AOAA revealed that the cofactor is the PLP form with the external aldimine linkage. The location of this oxime with the PLP, which forms in place of the native internal aldimine linkage of PLP of the native KAT‐I, is away from the position of the native internal aldimine, with the free Lys247 substantially retaining the orientation of the native structure. Tyr101, at the active site, was observed in two conformations in both structures.     

Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate.

Asp222 is an invariant residue in all known sequences of aspartate aminotransferases from a variety of sources and is located within a distance of strong ionic interaction with N(1) of the coenzyme, pyridoxal 5'-phosphate (PLP), or pyridoxamine 5'-phosphate (PMP). This residue of Escherichia coli aspartate aminotransferase was replaced by Ala, Asn, or Glu by site-directed mutagenesis. The PLP form of the mutant enzyme D222E showed pH-dependent spectral changes with a pKa value of 6.44 for the protonation of the internal aldimine bond, slightly lower than that (6.7) for the wild-type enzyme. In contrast, the internal aldimine bond in the D222A or D222N enzyme did not titrate over the pH range 5.3-9.5, and a 430-nm band attributed to the protonated aldimine persisted even at high pH. The binding affinity of the D222A and D222N enzymes for PMP decreased by 3 orders of magnitude as compared to that of the wild-type enzyme. Pre-steady-state half-transamination reactions of all the mutant enzymes with substrates exhibited anomalous progress curves comprising multiphasic exponential processes, which were accounted for by postulating several kinetically different enzyme species for both the PLP and PMP forms of each mutant enzyme. While the replacement of Asp222 by Glu yielded fairly active enzyme species, the replacement by Ala and Asn resulted in 8600- and 20,000-fold decreases, respectively, in the catalytic efficiency (kmax/Kd value for the most active species of each mutant enzyme) in the reactions of the PLP form with aspartate. In contrast, the catalytic efficiency of the PMP form of the D222A or D222N enzyme with 2-oxoglutarate was still retained at a level as high as 2-10% of that of the wild-type enzyme. The presteady-state reactions of these two mutant enzymes with [2-2H]aspartate revealed a deuterium isotope effect (kH/kD = 6.0) greater than that [kH/kD = 2.2; Kuramitsu, S., Hiromi, K., Hayashi, H., Morino, Y., & Kagamiyama, H. (1990) Biochemistry 29, 5469-5476] for the wild-type enzyme. These findings indicate that the presence of a negatively charged residue at position 222 is particularly critical for the withdrawal of the alpha-proton of the amino acid substrate and accelerates this rate-determining step by about 5 kcal.mol-1. Thus it is concluded that Asp222 serves as a protein ligand tethering the coenzyme in a productive mode within the active site and stabilizes the protonated N(1) of the coenzyme to strengthen the electron-withdrawing capacity of the coenzyme.     

Mechanism of substrate recognition and PLP-induced conformational changes in LL-diaminopimelate aminotransferase from Arabidopsis thaliana

LL-Diaminopimelate aminotransferase (LL-DAP-AT), a pyridoxal phosphate (PLP)-dependent enzyme in the lysine biosynthetic pathways of plants and Chlamydia, is a potential target for the development of herbicides or antibiotics. This homodimeric enzyme converts L-tetrahydrodipicolinic acid (THDP) directly to LL-DAP using L-glutamate as the source of the amino group. Earlier, we described the 3D structures of native and malate-bound LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT). Seven additional crystal structures of AtDAP-AT and its variants are reported here as part of an investigation into the mechanism of substrate recognition and catalysis. Two structures are of AtDAP-AT with reduced external aldimine analogues: N-(5'-phosphopyridoxyl)-L-glutamate (PLP-Glu) and N-(5'-phosphopyridoxyl)- LL-Diaminopimelate (PLP-DAP) bound in the active site. Surprisingly, they reveal that both L-glutamate and LL-DAP are recognized in a very similar fashion by the same sets of amino acid residues; both molecules adopt twisted V-shaped conformations. With both substrates, the alpha-carboxylates are bound in a salt bridge with Arg404, whereas the distal carboxylates are recognized via hydrogen bonds to the well-conserved side chains of Tyr37, Tyr125 and Lys129. The distal C(epsilon) amino group of LL-DAP is specifically recognized by several non-covalent interactions with residues from the other subunit (Asn309*, Tyr94*, Gly95*, and Glu97* (Amino acid designators followed by an asterisk (*) indicate that the residues originate in the other subunit of the dimer)) and by three bound water molecules. Two catalytically inactive variants of AtDAP-AT were created via site-directed mutagenesis of the active site lysine (K270N and K270Q). The structures of these variants permitted the observation of the unreduced external aldimines of PLP with L-glutamate and with LL-DAP in the active site, and revealed differences in the torsion angle about the PLP-substrate bond. Lastly, an apo-AtDAP-AT structure missing PLP revealed details of conformational changes induced by PLP binding and substrate entry into the active site.     

Mechanistic studies of 1-aminocyclopropane-1-carboxylate deaminase: characterization of an unusual pyridoxal 5'-phosphate-dependent reaction

1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that cleaves the cyclopropane ring of ACC, to give α-ketobutyric acid and ammonia as products. The cleavage of the C(α)-C(β) bond of an amino acid substrate is a rare event in PLP-dependent enzyme catalysis. Potential chemical mechanisms involving nucleophile- or acid-catalyzed cyclopropane ring opening have been proposed for the unusual transformation catalyzed by ACCD, but the actual mode of cyclopropane ring cleavage remains obscure. In this report, we aim to elucidate the mechanistic features of ACCD catalysis by investigating the kinetic properties of ACCD from Pseudomonas sp. ACP and several of its mutant enzymes. Our studies suggest that the pK(a) of the conserved active site residue, Tyr294, is lowered by a hydrogen bonding interaction with a second conserved residue, Tyr268. This allows Tyr294 to deprotonate the incoming amino group of ACC to initiate the aldimine exchange reaction between ACC and the PLP coenzyme and also likely helps to activate Tyr294 for a role as a nucleophile to attack and cleave the cyclopropane ring of the substrate. In addition, solvent kinetic isotope effect (KIE), proton inventory, and (13)C KIE studies of the wild type enzyme suggest that the C(α)-C(β) bond cleavage step in the chemical mechanism is at least partially rate-limiting under k(cat)/K(m) conditions and is likely preceded in the mechanism by a partially rate-limiting step involving the conversion of a stable gem-diamine intermediate into a reactive external aldimine intermediate that is poised for cyclopropane ring cleavage. When viewed within the context of previous mechanistic and structural studies of ACCD enzymes, our studies are most consistent with a mode of cyclopropane ring cleavage involving nucleophilic catalysis by Tyr294.     

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

Hydrogen sulfide (H₂S) 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, H₂S 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, H₂S is produced along with pyruvate and ammonia. To understand the H₂S‐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. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Strain is more important than electrostatic interaction in controlling the pKa of the catalytic group in aspartate aminotransferase

Systematic single and multiple replacement studies have been applied to Escherichia coli aspartate aminotransferase to probe the electrostatic effect of the two substrate-binding arginine residues, Arg292 and Arg386, and the structural effect of the pyridoxal 5'-phosphate-Asn194-Arg386 hydrogen-bond linkage system (PLP-N-R) on the pK(a) value of the Schiff base formed between pyridoxal 5'-phosphate (PLP) and Lys258. The electrostatic effects of the two arginine residues cannot be assessed by simple mutational studies of the residues. PLP-N-R lowers the pK(a) value of the PLP-Lys258 Schiff base by keeping it in the distorted conformation, which is unfavorable for protonation. Mutation of Arg386 eliminates its hydrogen bond with Asn194 and partially disrupts PLP-N-R, thereby relaxing the strain of the Schiff base. On the other hand, mutation of Arg292, the large domain residue that interacts with the small domain residue Asp15, makes the domain opening easier. Because PLP-N-R lies between the two domains, the domain opening increases the strain of the Schiff base. Therefore, the true electrostatic effects of Arg292 and Arg386 could be derived from mutational analysis of the enzyme in which PLP-N-R had been completely disrupted by the Asn194Ala mutation. Through the analyses, we could dissect the electrostatic and structural effects of the arginine mutations on the Schiff base pK(a). The positive charges of the two arginine residues and the PLP-N-R-mediated strain of the Schiff base lower the Schiff base pK(a) by 0.7 and 1.7, respectively. Thus, the electrostatic effect of the arginine residues is not as strong as has historically been thought, and this finding substantiates our recent finding that the imine-pyridine torsion of the Schiff base is the primary determinant (2.8 unit decrease) of the extremely low pK(a) value of the Schiff base [Hayashi, H., Mizuguchi, H., and Kagamiyama, H. (1998) Biochemistry 37, 15076-15085].