Preliminary x-ray data on crystals of mandelate racemase.

Neidhart, David J.; Powers, Vincent M.; Kenyon, George L.; Tsou, Amy Y.; Ransom, Stephen C.; Gerlt, J.A.; Petsko, Gregory A.

doi:10.1016/s0021-9258(19)76534-x

Cited by 12 publications

(2 citation statements)

References 15 publications

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“…Much mechanistic information about the racemization reaction was obtained from analyzing the 3-D structure of mandelate racemase (EC 5.1.2.2, Pseudomonas putida, 38.5 kDa) which has beeen crystallized by Neidhart et al (1988).…”

Section: Discussionmentioning

confidence: 99%

Gramicidin S synthetase 1 (phenylalanine racemase), a prototype of amino acid racemases containing the cofactor 4'-phosphopantetheine

Stein

Kluge²,

Vater³

et al. 1995

Biochemistry

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The biosynthesis of the decapeptide antibiotic gramicidin S in Bacillus brevis ATCC 9999 is catalyzed by a multienzyme system consisting of two multifunctional proteins, gramicidin S synthetase 1 and 2, encoded by the grsA and grsB genes, respectively. Gramicidin S synthetase 1 (phenylalanine racemase, EC 5.1.1.11, GS1) racemizes phenylalanine in the thioester-bound stage. The amount of 4'-phosphopantetheine liberated from highly purified GS1 was determined microbiologically using Lacto-bacillus plantarum as the test organism. It matches exactly with the amount of L-[14C]phenylalanine covalently incorporated by GS1 as thioester. The reaction center of GS1 for L-phenylalanine thiolation and racemization was labeled with [3H]iodoacetic acid. After tryptic fragmentation of the 3H-carboxymethylated enzyme, the active site peptide for thioester binding and racemization of phenylalanine was isolated in pure form by multistep methodology and investigated by sequence, amino acid, and mass spectrometric analysis. A 4'-phosphopantetheine carrier was found to be attached to the active site serine of the consensus motif LGGDSI forming the thiolation site of phenylalanine. These specific properties establish GS1 as a prototype of amino acid racemases using 4'-phosphopantetheine as a cofactor and yield further evidence that multiple Pan carriers are involved in gramicidin S formation. Our results are strong evidence for the "multiple carrier model" as a new concept of nonribosomal peptide biosynthesis at protein templates as recently proposed [Stein, T., et al. (1994) FEBS Lett. 340, 39-44].

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Section: Discussionmentioning

confidence: 99%

Gramicidin S synthetase 1 (phenylalanine racemase), a prototype of amino acid racemases containing the cofactor 4'-phosphopantetheine

Stein

Kluge²,

Vater³

et al. 1995

Biochemistry

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“…The structural studies described herein permit the identification of likely catalytic residues in the active site of MR, including the two acid/base catalysts, and also establish the general disposition of the substrate in the active site. We have previously reported two crystal forms of MR (Neidhart et al, 1988); however, the crystal structure of the enzyme has now been solved with use of a newly discovered crystal form that diffracts to high resolution (<2.0 Á) and contains a single subunit of MR in the asymmetric unit. We report the conditions used to prepare this crystal form and the methods used to solve the X-ray crystal structure by multiple isomorphous replacement at 3.0-Á resolution.…”

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confidence: 99%

Mechanism of the reaction catalyzed by mandelate racemase. 2. Crystal structure of mandelate racemase at 2.5-.ANG. resolution: identification of the active site and possible catalytic residues

et al. 1991

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The crystal structure of mandelate racemase (MR) has been solved at 3.0-A resolution by multiple isomorphous replacement and subsequently refined against X-ray diffraction data to 2.5-A resolution by use of both molecular dynamics refinement (XPLOR) and restrained least-squares refinement (PROLSQ). The current crystallographic R-factor for this structure is 18.3%. MR is composed of two major structural domains and a third, smaller, C-terminal domain. The N-terminal domain has an alpha + beta topology consisting of a three-stranded antiparallel beta-sheet followed by an antiparallel four alpha-helix bundle. The central domain is a singly wound parallel alpha/beta-barrel composed of eight central strands of beta-sheet and seven alpha-helices. The C-terminal domain consists of an irregular L-shaped loop with several short sections of antiparallel beta-sheet and two short alpha-helices. This C-terminal domain partially covers the junction between the major domains and occupies a region of the central domain that is filled by an eight alpha-helix in all other known parallel alpha/beta-barrels except for the barrel domain in muconate lactonizing enzyme (MLE) [Goldman, A., Ollis, D. L., & Steitz, T. A. (1987) J. Mol. Biol. 194, 143] whose overall polypeptide fold and amino acid sequence are strikingly similar to those of MR [Neidhart, D. J., Kenyon, G. L., Gerlt, J. A., & Petsko, G. A. (1990) Nature 347, 692]. In addition, the crystal structure reveals that, like MLE, MR is tightly packed as an octamer of identical subunits. The active site of MR is located between the two major domains, at the C-terminal ends of the beta-strands in the alpha/beta-barrel domain. The catalytically essential divalent metal ion is ligated by three side-chain carboxyl groups contributed by residues of the central beta-sheet. A model of a productive substrate complex of MR has been constructed on the basis of difference Fourier analysis at 3.5-A resolution of a complex between MR and (R,S)-p-iodomandelate, permitting identification of residues that may participate in substrate binding and catalysis. The ionizable groups of both Lys 166 and His 297 are positioned to interact with the chiral center of substrate, suggesting that both of these residues may function as acid/base catalysts.(ABSTRACT TRUNCATED AT 400 WORDS)

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