1MNS

ON THE ROLE OF LYSINE 166 IN THE MECHANISM OF MANDELATE RACEMASE FROM PSEUDOMONAS PUTIDA: MECHANISTIC AND CRYSTALLOGRAPHIC EVIDENCE FOR STEREOSPECIFIC ALKYLATION BY (R)-ALPHA-PHENYLGLYCIDATE

Summary for 1MNS

• Entry DOI: 10.2210/pdb1mns/pdb
• Descriptor: MANDELATE RACEMASE, MAGNESIUM ION, ATROLACTIC ACID (2-PHENYL-LACTIC ACID), ... (4 entities in total)
• Functional Keywords: racemase
• Biological source: Pseudomonas putida
• Total number of polymer chains: 1
• Total formula weight: 38574.80

Authors

Neidhart, D.J.,Landro, J.A.,Kozarich, J.W. (deposition date: 1993-07-06, release date: 1993-10-31, Last modification date: 2024-10-30)

Primary citation

Landro, J.A.,Gerlt, J.A.,Kozarich, J.W.,Koo, C.W.,Shah, V.J.,Kenyon, G.L.,Neidhart, D.J.,Fujita, S.,Petsko, G.A.
The role of lysine 166 in the mechanism of mandelate racemase from Pseudomonas putida: mechanistic and crystallographic evidence for stereospecific alkylation by (R)-alpha-phenylglycidate.
Biochemistry, 33:635-643, 1994

PubMed Abstract: The mechanism of irreversible inactivation of mandelate racemase (MR) from Pseudomonas putida by alpha-phenylglycidate (alpha PGA) has been investigated stereochemically and crystallographically. The (R) and (S) enantiomers of alpha PGA were synthesized in high enantiomeric excess (81% ee and 83% ee, respectively) using Sharpless epoxidation chemistry. (R)-alpha PGA was determined to be a stereospecific and stoichiometric irreversible inactivator of MR. (S)-alpha PGA does not inactivate MR and appears to bind noncovalently to the active site of MR with less affinity than that of (R)-alpha PGA. The X-ray crystal structure (2.0-A resolution) of MR inactivated by (R)-alpha PGA revealed the presence of a covalent adduct formed by nucleophilic attack of the epsilon-amino group of Lys 166 on the distal carbon on the epoxide ring of (R)-alpha PGA. The proximity of the alpha-proton of (S)-mandelate to Lys 166 [configurationally equivalent to (R)-alpha PGA] was corroborated by the crystal structure (2.1-A resolution) of MR complexed with the substrate analog/competitive inhibitor, (S)-atrolactate [(S)-alpha-methylmandelate]. These results support the proposal that Lys 166 is the polyvalent acid/base responsible for proton transfers on the (S) face of mandelate. In addition, the high-resolution structures also provide insight into the probable interactions of mandelate with the essential Mg2+ and functional groups in the active site.

PubMed: 8292591
DOI: 10.1021/bi00169a003

Experimental method

X-RAY DIFFRACTION (2 Å)