Bacterial N-succinyl-L-diaminopimelic acid desuccinylase. Purification, partial characterization, and substrate specificity.

Lin, Yukang; Myhrman, R.; Schrag, Michael; Gelb, Michael H.

doi:10.1016/s0021-9258(19)77924-1

Cited by 41 publications

(19 citation statements)

References 21 publications

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“…L-lysine biosynthesis in E. coli occurs exclusively through the succinylase pathway (Gilvarg & Weinberger, 1970) although other bacteria use either the biochemically equivalent acetylase pathway or the dehydrogenase shunt. All of the enzymes in the succinylase pathway, Scheme 1, have been purified and partially characterized from E. coli, including the synthase (dapA; Shedlarski & Gilvarg, 1970), reductase (dapB; Tamir & Gilvarg, 1974), A-succinylase 0dapC; Simms et al, 1984), transaminase (dapD; Peterkofsky & Gilvarg, 1961), desuccinylase (dapE; Lin et al, 1988), epimerase (dapF; Wiseman & Nichols, 1984), and decarboxylase (lysA; Kelly & White, 1965). While some of the genes encoding these enzymes appear to be clustered in Corynebacterium glutamicum (Cremer et al, 1991) and E. coli (Richaud et al, 1986), others are scattered throughout the E. coli chromosome (Bachman, 1990).…”

Section: Discussionmentioning

confidence: 99%

Expression, Purification, and Characterization of Escherichia coli Dihydrodipicolinate Reductase

Reddy¹,

Sacchettini²,

Blanchard³

1995

Biochemistry

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Dihydrodipicolinate reductase catalyzes the NAD(P)H-dependent reduction of the carbon-carbon double bond of the alpha,beta-unsaturated cyclic imine dihydrodipicolinate to form the cyclic imine tetrahydrodipicolinate. The enzyme is a component of the bacterial biosynthetic pathway forming L-lysine and diaminopimelate from L-aspartate. The gene encoding dihydrodipicolinate reductase, dapB, has been cloned and sequenced from Escherichia coli (Bouvier et al., 1984), and we have used this sequence information to generate an expression vector containing the dapB gene. Expression and purification of dihydrodipicolinate reductase to homogeneity have allowed us to characterize the kinetics, stereochemistry, and chemical mechanism of the enzymatic reaction. The kinetic mechanism is ordered, with reduced nucleotide binding preceding dihydrodipicolinate binding and presumably with tetrahydrodipicolinate dissociating prior to oxidized nucleotide release. The enzyme has a unique nucleotide specificity, with NADH being twice as effective as NADPH as a reductant. The enzyme catalyzes the stereospecific transfer of the 4R hydrogen atom of the reduced nucleotide, as a hydride ion, to the 4 position of dihydrodipicolinate. These results allow us to propose a chemical mechanism for the reaction catalyzed by dihydrodipicolinate reductase involving hydride transfer to the beta carbon of the unsaturated imine. The resonance-stabilized C3 carbanion is protonated to generate the reduced product, tetrahydrodipicolinate.

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Section: Discussionmentioning

confidence: 99%

Expression, Purification, and Characterization of Escherichia coli Dihydrodipicolinate Reductase

Reddy¹,

Sacchettini²,

Blanchard³

1995

Biochemistry

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“…SDAP was synthesized using the procedure described by Lin et al, providing an overall yield of 41%. The specific activities of all Hi DapE and Nm DapE proteins were determined using a 50/50 mixture of d , d - and l , l -SDAP as the substrate in 50 mM phosphate buffer (PP i ) or 50 mM HEPES (pH 7.5), as previously described .…”

Section: Methodsmentioning

confidence: 99%

Structural Evidence of a Major Conformational Change Triggered by Substrate Binding in DapE Enzymes: Impact on the Catalytic Mechanism

et al. 2018

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The X-ray crystal structure of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae (HiDapE) bound by the products of hydrolysis, succinic acid and l,l-DAP, was determined at 1.95 Å. Surprisingly, the structure bound to the products revealed that HiDapE undergoes a significant conformational change in which the catalytic domain rotates ∼50° and shifts ∼10.1 Å (as measured at the position of the Zn atoms) relative to the dimerization domain. This heretofore unobserved closed conformation revealed significant movements within the catalytic domain compared to that of wild-type HiDapE, which results in effectively closing off access to the dinuclear Zn(II) active site with the succinate carboxylate moiety bridging the dinculear Zn(II) cluster in a μ-1,3 fashion forming a bis(μ-carboxylato)dizinc(II) core with a Zn-Zn distance of 3.8 Å. Surprisingly, His194.B, which is located on the dimerization domain of the opposing chain ∼10.1 Å from the dinuclear Zn(II) active site, forms a hydrogen bond (2.9 Å) with the oxygen atom of succinic acid bound to Zn2, forming an oxyanion hole. As the closed structure forms upon substrate binding, the movement of His194.B by more than ∼10 Å is critical, based on site-directed mutagenesis data, for activation of the scissile carbonyl carbon of the substrate for nucleophilic attack by a hydroxide nucleophile. Employing the HiDapE product-bound structure as the starting point, a reverse engineering approach called product-based transition-state modeling provided structural models for each major catalytic step. These data provide insight into the catalytic reaction mechanism and also the future design of new, potent inhibitors of DapE enzymes.

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“…Studies with metal-free PGGT-I were carried out in buffer C (30 mM HEPES-NaOH, 5 mM DTT, pH 7.7). Solutions of buffer and peptide substrates were rendered metal-free by extraction with dithizone (Lin et al, 1988). Stock solutions of metals were made by dissolving the solids in Milli-Q water.…”

Section: Methodsmentioning

confidence: 99%

Mammalian Protein Geranylgeranyltransferase-I: Substrate Specificity, Kinetic Mechanism, Metal Requirements, and Affinity Labeling

Yokoyama¹,

McGeady²,

Gelb³

1995

Biochemistry

122

125

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Protein geranylgeranyltransferase-I (PGGT-I) catalyzes the transfer of the 20-carbon prenyl group from geranylgeranyl pyrophosphate to the cysteine residue near the C-termini of a variety of eukaryotic proteins. Kinetic analysis of homogenous PGGT-I from bovine brain reveals that the reaction follows a sequential pathway in which either prenyl donor or acceptor can bind first to the enzyme and that the reaction operates at steady-state rather than at rapid equilibrium. Substrate inhibition by prenyl acceptor but not by prenyl donor suggests that geranylgeranyl pyrophosphate binding first to free enzyme is the kinetically preferred pathway. This is supported by isotope trapping experiments which show that the ternary complex goes on to products faster than the release of geranylgeranyl pyrophosphate from the complex. The KM for the interaction of geranylgeranyl pyrophosphate with PGGT-I is markedly affected by the structure of the prenyl acceptor bound to the enzyme. A detailed analysis of the substrate specificity of PGGT-I reveals that peptides which contain a C-terminal leucine are preferred (kcat/KM = 1-5 x 10(5) M-1 s-1) to those that end in serine (kcat/KM = 2-4 x 10(3) M-1 s-1) or phenylalanine (kcat/KM = 0.5 x 10(3) M-1 s-1). PGGT-I also catalyzes the farnesylation of peptides that have a C-terminal leucine; kcat for farnesylation and KM for farnesyl pyrophosphate are similar to those for geranylgeranylation, but the KM for the peptide is 30-fold higher. Geranyl pyrophosphate is utilized by PGGT-I but is a poor substrate. Optimal activity of PGGT-I is obtained in the presence of micromolar amounts of Zn2+ and mM amounts of Mg2+. Mn2+ or Cd2+ but not Co2+ can substitute for Zn2+ and for Mg2+. Metals are not required for tight-binding of geranylgeranyl pyrophosphate to PGGT-I, and the measured dissociation equilibrium constant for this binary complex is 16 nM. Photoaffinity analogues of geranylgeranyl pyrophosphate and farnesyl pyrophosphate were prepared and shown to exclusively label the beta-subunit. The implication of the results for the substrate specificity of protein prenylation in cells is briefly discussed.

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Bacterial N-succinyl-L-diaminopimelic acid desuccinylase. Purification, partial characterization, and substrate specificity.

Cited by 41 publications

References 21 publications

Expression, Purification, and Characterization of Escherichia coli Dihydrodipicolinate Reductase

Expression, Purification, and Characterization of Escherichia coli Dihydrodipicolinate Reductase

Structural Evidence of a Major Conformational Change Triggered by Substrate Binding in DapE Enzymes: Impact on the Catalytic Mechanism

Mammalian Protein Geranylgeranyltransferase-I: Substrate Specificity, Kinetic Mechanism, Metal Requirements, and Affinity Labeling

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