title: Conserved main-chain peptide distortions: a proposed role for Ile203 in catalysis by dihydrodipicolinate synthase.
abstract: In recent years, dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) has received considerable attention from a mechanistic and structural viewpoint. DHDPS catalyzes the reaction of (S)-aspartate-beta-semialdehyde with pyruvate, which is bound via a Schiff base to a conserved active-site lysine (Lys161 in the enzyme from Escherichia coli). To probe the mechanism of DHDPS, we have studied the inhibition of E. coli DHDPS by the substrate analog, beta-hydroxypyruvate. The K (i) was determined to be 0.21 (+/-0.02) mM, similar to that of the allosteric inhibitor, (S)-lysine, and beta-hydroxypyruvate was observed to cause time-dependent inhibition. The inhibitory reaction with beta-hydroxypyruvate could be qualitatively followed by mass spectrometry, which showed initial noncovalent adduct formation, followed by the slow formation of the covalent adduct. It is unclear whether beta-hydroxypyruvate plays a role in regulating the biosynthesis of meso-diaminopimelate and (S)-lysine in E. coli, although we note that it is present in vivo. The crystal structure of DHDPS complexed with beta-hydroxypyruvate was solved. The active site clearly showed the presence of the inhibitor covalently bound to the Lys161. Interestingly, the hydroxyl group of beta-hydroxypyruvate was hydrogen-bonded to the main-chain carbonyl of Ile203. This provides insight into the possible catalytic role played by this peptide unit, which has a highly strained torsion angle (omega approximately 201 degrees ). A survey of the known DHDPS structures from other organisms shows this distortion to be a highly conserved feature of the DHDPS active site, and we propose that this peptide unit plays a critical role in catalysis.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia. rdobson@unimelb.edu.au
authors.forename: Renwick C J
authors.initials: RC
authors.lastname: Dobson
authors.forename: Michael D W
authors.initials: MD
authors.lastname: Griffin
authors.forename: Sean R A
authors.initials: SR
authors.lastname: Devenish
authors.forename: F Grant
authors.initials: FG
authors.lastname: Pearce
authors.forename: Craig A
authors.initials: CA
authors.lastname: Hutton
authors.forename: Juliet A
authors.initials: JA
authors.lastname: Gerrard
authors.forename: Geoffrey B
authors.initials: GB
authors.lastname: Jameson
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Perugini
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D007532:Isoleucine / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000096:biosynthesis / Q000378:metabolism*; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011773:Pyruvates / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D020124:Thermotoga maritima / Q000201:enzymology
chemical_list: D010455:Peptides; D011773:Pyruvates; D007532:Isoleucine; C012375:hydroxypyruvic acid; D006836:Hydro-Lyases; C020040:4-hydroxy-tetrahydrodipicolinate synthase; D008239:Lysine
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