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@ARTICLE{Bruning:93808,
      author       = {Bruning, M. and Berheide, M. and Meyer, D. and Golbik, R.
                      and Bartunik, H. D. and Liese, A. and Tittmann, K. and DESY},
      title        = {{S}tructural and kinetic studies on native intermediates
                      and an intermediate analogue in benzoylformate decarboxylase
                      reveal a least motion mechanism with an unprecedented
                      short-lived predecarboxylation intermediate},
      journal      = {Biochemistry},
      volume       = {48},
      issn         = {0006-2960},
      address      = {Columbus, Ohio},
      publisher    = {American Chemical Society},
      reportid     = {PHPPUBDB-12176},
      pages        = {3258-3268},
      year         = {2009},
      note         = {(c) American Chemical Society},
      abstract     = {The thiamin diphosphate- (ThDP-) dependent enzyme
                      benzoylformate decarboxylase (BFDC) catalyzes the
                      nonoxidative decarboxylation of benzoylformic acid to
                      benzaldehyde and carbon dioxide. To date, no structural
                      information for a cofactor-bound reaction intermediate in
                      BFDC is available. For kinetic analysis, a chromophoric
                      substrate analogue was employed that produces various
                      absorbing intermediates during turnover but is a poor
                      substrate with a 10(4)-fold compromised kcat. Here, we have
                      analyzed the steady-state distribution of native
                      intermediates by a combined chemical quench/1H NMR
                      spectroscopic approach and estimated the net rate constants
                      of elementary catalytic steps. At substrate saturation,
                      carbonyl addition of the substrate to the cofactor (k'
                      approximately 500 s-1 at 30 degrees C) and elimination of
                      benzaldehyde (k' approximately 2.400 s-1) were found to be
                      partially rate-determining for catalysis, whereas
                      decarboxylation of the transient 2-mandelyl-ThDP
                      intermediate is 1 order of magnitude faster with k'
                      approximately 16.000 s-1, the largest rate constant of
                      decarboxylation in any thiamin enzyme characterized so far.
                      The X-ray structure of a predecarboxylation intermediate
                      analogue was determined to 1.6 A after cocrystallization of
                      BFDC from Pseudomonas putida with benzoylphosphonic acid
                      methyl ester. In contrast to the free acid, for which
                      irreversible phosphorylation of active center Ser26 was
                      reported, the methyl ester forms a covalent adduct with ThDP
                      with a similar configuration at C2alpha as observed for
                      other thiamin enzymes. The C2-C2alpha bond of the
                      intermediate analogue is out of plane by 7degrees,
                      indicating strain. The phosphonate part of the adduct forms
                      hydrogen bonds with Ser26 and His281, and the 1-OH group is
                      held in place by interactions with His70 and the 4'-amino
                      group of ThDP. The phenyl ring accommodates in a hydrophobic
                      pocket formed by Phe464, Phe397, Leu109, and Leu403. A
                      comparison with the previously determined structure of BFDC
                      in noncovalent complex with the inhibitor (R)-mandelate
                      suggests a least motion mechanism. Binding of
                      benzoylphosphonic acid methyl ester to BFDC was further
                      characterized by CD spectroscopy and stopped-flow kinetics,
                      indicating a two-step binding mechanism with a 200-fold
                      slower carbonyl addition to ThDP than determined for
                      benzoylformic acid, in line with the observed slight
                      structural reorganization of Phe464 due to steric clashes
                      with the phosphonate moiety.},
      keywords     = {Bacterial Proteins: chemistry / Bacterial Proteins:
                      metabolism / Carboxy-Lyases: chemistry / Carboxy-Lyases:
                      metabolism / Catalysis / Crystallography, X-Ray /
                      Decarboxylation / Kinetics / Magnetic Resonance Spectroscopy
                      / Pseudomonas putida: enzymology / Structure-Activity
                      Relationship / Substrate Specificity / Thermodynamics / Time
                      Factors / Bacterial Proteins (NLM Chemicals) /
                      Carboxy-Lyases (NLM Chemicals) / benzoylformate
                      decarboxylase (NLM Chemicals)},
      cin          = {HASYLAB(-2012) / MPG(-2012)},
      ddc          = {570},
      cid          = {$I:(DE-H253)HASYLAB_-2012_-20130307$ /
                      $I:(DE-H253)MPG_-2012_-20120307$},
      pnm          = {Facility (machine) DORIS/PETRA (POF1-DORIS-PETRA-20130405)},
      pid          = {G:(DE-H253)POF1-DORIS-PETRA-20130405},
      experiment   = {EXP:(DE-H253)DORISIII(machine)-20150101},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:19182954},
      UT           = {WOS:000265170200003},
      doi          = {10.1021/bi801957d},
      url          = {https://bib-pubdb1.desy.de/record/93808},
}