Structural and Mechanistic Analysis of Two Refined Crystal Structures of the Pyridoxal Phosphate-dependent Enzyme Dialkylglycine Decarboxylase

Toney, Michael D.; Hohenester, Erhard; Keller, John W.; Jansonius, Johan N.

doi:10.1006/jmbi.1994.0014

Cited by 109 publications

(140 citation statements)

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“…The crystal structures of four different alkali metal liganded DGD forms have been reported (4)(5)(6). In solution, two coexisting, slowly interconverting conformations of homogeneously K + -liganded DGD were demonstrated (3).…”

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Rapid Kinetic and Isotopic Studies on Dialkylglycine Decarboxylase

et al. 2001

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The two half-reactions of the pyridoxal 5′-phosphate (PLP)-dependent enzyme dialkylglycine decarboxylase (DGD) were studied individually by multiwavelength stopped-flow spectroscopy. Biphasic behavior was found for the reactions of DGD-PLP, consistent with two coexisting conformations observed in steady-state kinetics [Zhou, X., and Toney, M. D. (1998) Biochemistry 37, 5761-5769]. The halfreaction kinetic parameters depend on alkali metal ion size in a manner similar to that observed for steadystate kinetic parameters. The fast phase maximal rate constant for the 2-aminoisobutyrate (AIB) decarboxylation half-reaction with the potassium form of DGD-PLP is 25 s -1 , while that for the transamination half-reaction between DGD-PMP and pyruvate is 75 s -1 . The maximal rate constant for the transamination half-reaction of the potassium form of DGD-PLP with L-alanine is 24 s -1 . The spectral data indicate that external aldimine formation with either AIB or L-alanine and DGD-PLP is a rapid equilibrium process, as is ketimine formation from DGD-PMP and pyruvate. Absorption ascribable to the quinonoid intermediate is not observed in the AIB decarboxylation half-reaction, but is observed in the dead-time of the stopped-flow in the L-alanine transamination half-reaction. The [1-13 C]AIB kinetic isotope effect (KIE) on k cat for the steady-state reaction is 1.043 ( 0.003, while a value of 1.042 ( 0.009 was measured for the AIB half-reaction. The secondary KIE measured for the AIB decarboxylation halfreaction with [C4′-2 H]PLP is 0.92 ( 0.02. The primary [2-2 H]-L-alanine KIE on the transamination halfreaction is unity. Small but significant solvent KIEs are observed on k cat and k cat /K M for both substrates, and the proton inventories are linear in each case. NMR measurements of C2-H washout vs product formation give ratios of 105 and 14 with L-alanine and isopropylamine as substrates, respectively. These results support a rate-limiting, concerted CR-decarboxylation/C4′-protonation mechanism for the AIB decarboxylation reaction, and rapid equilibrium quinonoid formation followed by rate-limiting protonation to the ketimine intermediate for the L-alanine transamination half-reaction. Energy profiles for the two half-reactions are constructed.Dialkylglycine decarboxylase (DGD) 1 is an intriguing PLP-dependent enzyme because it catalyzes both transamination and decarboxylation reactions in its normal catalytic cycle (Scheme 1). The enzyme was isolated from a soil bacterium (1) and transfers the fixed nitrogen from small 2,2-dialkylglycines, which are common in peptide antibiotics (2), onto pyruvate to give L-alanine. The decarboxylation half-reaction is highly specific for oxidative decarboxylation. Thus, the issue of reaction specificity has two main facets with this enzyme: control of decarboxylation vs transamination, and control of oxidative vs nonoxidative decarboxylation. The latter is addressed in this work.Previous work has shown that DGD activity is dependent on alkali metal ions; potassium activates it w...

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Rapid Kinetic and Isotopic Studies on Dialkylglycine Decarboxylase

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“…With regards to the co-factor-binding pocket in the active site of PLP-dependent enzymes, details of how the different proteins interact with the co-enzyme have been well-defined only in the cases for which high-resolution crystal structures have been determined (Kirsch et al, 1984;Hyde et al, 1988;McPhalen et al, 1992;Antson et al, 1993;Momany et al, 1995b;Toney et al, 1995). In particular, a glycine-rich loop has been recognized at the co-factor binding site in those enzymes for which crystal structures are available and has also been identified through site-directed mutagenesis in threonine and D-serine dehydratases (Dana et al, 1987;Marceau et al, 1990) and more recently in aminolevulinate synthase (Gong et al, 1995(Gong et al, , 1996.…”

Section: Discussionmentioning

confidence: 99%

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation

et al. 1997

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Cysteine 111 in Dopa decarboxylase (DDC) has been replaced by alanine or serine by site-directed mutagenesis. Compared to the wild-type enzyme, the resultant C111A and C111 S mutant enzymes exhibit kc,, values of about 50% and 15%, respectively, at pH 6.8, while the K, values remain relatively unaltered for L-3,4-dihydroxyphenylalanine (L-Dopa) and L-5-hydroxytryptophan (LJ-HTP). While a significant decrease of the 280 nm optically active band present in the wild type is observed in mutant DDCs, their visible co-enzyme absorption and CD spectra are similar to those of the wild type. With respect to the wild type, the Cys-1 1 ]-+Ala mutant displays a reduced affinity for pyridoxal 5"phosphate (PLP), slower kinetics of reconstitution to holoenzyme, a decreased ability to anchor the external aldimine formed between D-Dopa and the bound co-enzyme, and a decreased efficiency of energy transfer between tryptophan residue(s) and reduced PLP. Values of pK, and pKb for the groups involved in catalysis were determined for the wild-type and the C l l l A mutant enzymes. The mutant showed a decrease in both pK values by about 1 pH unit, resulting in a shift of the pH of the maximum velocity from 7.2 (wild-type) to 6.2 (mutant). This change in maximum velocity is mirrored by a similar shift in the spectrophotometrically determined pK value of the 420 -+ 390 nm transition of the external aldimine. These results demonstrate that the sulfhydryl group of Cys-1 1 1 is catalytically nonessential and provide strong support for previous suggestion that this residue is located at or near the PLP binding site (Dominici P, Maras B, Mei G, Bom Voltattorni C. 1991. Eur JBiochern 201:393-397). Moreover, our findings provide evidence that Cys-111 has a structural role in PLP binding and suggest that this residue is required for maintenance of proper active-site conformation.Keywords: active site; decarboxylase; pyridoxal 5"phosphate; site-directed mutagenesis The versatility of pyridoxal 5"phosphate (PLP) as a co-factor is demonstrated by the wide variety of reactions that enzymes dependent upon it catalyze. Although detailed catalytic mechanisms and information correlating structure and function have been reported for many PLP dependent enzymes, such information is conspicuously lacking for PLP dependent decarboxylases.Even though aromatic amino acid decarboxylases have been cloned from a number of mammalian (Ichinose et al., 1989;Tanaka et al., 1989;Kang & Joh, 1990;Taketoshi et al., 1990), insect (Eveleth et al., 1986, and plant sources (DeLuca et al., 1989;Kawalleck et al., 1993; Facchini & DeLuca, 1994)

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“…The crystals of the DGD-PMP enzyme were prepared by soaking WT DGD-PLP crystals obtained as previously described (14) in a stabilizing buffer containing 30% PEG4000, 15 mM MES-KOH (pH 6.4), and 1 mM AIB for 30 min to convert PLP to PMP in the enzyme active site. These crystals were then soaked in a buffer containing 30% PEG4000, 15% MES-KOH (pH 6.4), and 10 mM PMP for 2 h, and finally soaked in a cryo-protecting solution containing 30% PEG4000, 15 mM MES-KOH (pH 6.4), 10 mM AIB, and 26% glycerol for 1 day.…”

Section: Methodsmentioning

confidence: 99%

“…Although the omit electron density map clearly indicates the existence of a water molecule filling the space created by the Q52A mutation ( Figure 3B), the temperature factor of this water molecule (43.9) is significantly higher than the average temperature factor of the peptide (25.8) or solvent molecules (19.4). The Q52A mutation, which eliminates a hydrogen bond that normally helps to hold Y301 in place (14), and the mobility of the water molecule taking the place of the Q52 side chain lead to a relatively high mobility of the Y301 side chain (average B ) 37). The superimposition of the Q52A dimer and WT dimer gives a pairwise rms deviation of 0.37 Å with no significant differences other than the site of mutation.…”

Section: X-ray Crystallographic Strctures Of Q52a-plp and Wt Dgd-pmpmentioning

confidence: 99%

“…A functional active site model based on the unusual reaction specificity of DGD and stereoelectronic control of reaction specificity has been proposed (14) and validated (11). In this model, there are three subsites, the A subsite, near Q52 and K272, the stereoelectronically activated position in which all bond breaking and making occur; the B subsite, near R406 and S215, which can bind a carboxylate; and the C subsite, near M141 ( Figure 1).…”

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Role of Q52 in Catalysis of Decarboxylation and Transamination in Dialkylglycine Decarboxylase

et al. 2005

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Dialkylglycine decarboxylase (DGD) is a pyridoxal phosphate dependent enzyme that catalyzes both decarboxylation and transamination in its normal catalytic cycle. DGD uses stereoelectronic effects to control its unusual reaction specificity. X-ray crystallographic structures of DGD suggest that Q52 is important in maintaining the substrate carboxylate in a stereoelectronically activated position. Here, the X-ray structures of the Q52A mutant and the wild type (WT) DGD-PMP enzymes are presented, as is the analysis of steady-state and half-reaction kinetics of three Q52 mutants (Q52A, Q52I, and Q52E). As expected if stereoelectronic effects are important to catalysis, the steady-state rate of decarboxylation for all three mutants has decreased significantly compared to that of WT. Q52A exhibits an ∼85-fold decrease in k cat relative to that of WT. The rate of the decarboxylation half-reaction decreases ∼10 5 -fold in Q52I and ∼10 4 -fold in Q52E compared to that of WT. Transamination half-reaction kinetics show that Q52A and Q52I have greatly reduced rates compared to that of WT and are seriously impaired in pyridoxamine phosphate (PMP) binding, with K PMP at least 50-100-fold greater than that of WT. The larger effect on the rate of L-alanine transamination than of pyruvate transamination in these mutants suggests that the rate decrease is the result of selective destabilization of the PMP form of the enzyme in these mutants. Q52E exhibits near-WT rates for transamination of both pyruvate and L-alanine. Substrate binding has been greatly weakened in Q52E with apparent dissociation constants at least 100-fold greater than that of WT. The rate of decarboxylation in Q52E allows the energetic contribution of stereoelectronic effects, ∆G stereoelectronic , to be estimated to be -7.3 kcal/mol for DGD.Pyridoxal phosphate (PLP) 1 dependent enzymes constitute a large and well-characterized group of enzymes, catalyzing many different types of chemistry at the R-, -, and γ-carbons of amine and amino acid substrates. Despite the diversity of chemistry catalyzed by PLP dependent enzymes as a group, individual enzymes exhibit remarkably high reaction specificities. In contrast, model studies show that PLP alone is capable of nonenzymatically catalyzing multiple reaction types with a given substrate. For example, reaction of PLP with serine results in transamination, -elimination, and retroaldol cleavage (1). As an explanation for the high enzymatic versus nonenzymatic specificity, it was proposed by Dunathan (2) that PLP dependent enzymes use stereoelectronic effects to control reaction specificity. He proposed that the scissile bond is aligned parallel to the p orbitals of the extended π system of the aldimine, providing maximal orbital overlap and resonance stabilization in the transition state, therefore accelerating the rate of bond cleavage for the activated bond compared to the other bonds to C R .Stereoelectronic effects have been investigated in simple organic systems both experimentally and computationally (...

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Structural and Mechanistic Analysis of Two Refined Crystal Structures of the Pyridoxal Phosphate-dependent Enzyme Dialkylglycine Decarboxylase

Cited by 109 publications

References 71 publications

Rapid Kinetic and Isotopic Studies on Dialkylglycine Decarboxylase

Rapid Kinetic and Isotopic Studies on Dialkylglycine Decarboxylase

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation

Role of Q52 in Catalysis of Decarboxylation and Transamination in Dialkylglycine Decarboxylase

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