1sze › Citations

Altered substrate specificity in flavocytochrome b2: structural insights into the mechanism of L-lactate dehydrogenation.

Mowat CG, Wehenkel A, Green AJ, Walkinshaw MD, Reid GA, Chapman SK
Biochemistry 43 9519-26 (2004)
Related entries: 1szf, 1szg

Cited: 21 times
EuropePMC logo PMID: 15260495

Abstract

Flavocytochrome b(2) from Saccharomyces cerevisiae is a l-lactate/cytochrome c oxidoreductase belonging to a large family of 2-hydroxyacid-dependent flavoenzymes. The crystal structure of the enzyme, with pyruvate bound at the active site, has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863]. The authors indicate that the methyl group of pyruvate is in close contact with Ala198 and Leu230. These two residues are not well-conserved throughout the family of (S)-2-hydroxy acid oxidases/dehydrogenases. Thus, to probe substrate specificity in flavocytochrome b(2), these residues have been substituted by glycine and alanine, respectively. Kinetic studies on the L230A mutant enzyme and the A198G/L230A double mutant enzyme indicate a change in substrate selectivity for the enzyme toward larger (S)-2-hydroxy acids. In particular, the L230A enzyme is more efficient at utilizing (S)-2-hydroxyoctanoate by a factor of 40 as compared to the wild-type enzyme [Daff, S., Manson, F. D. C., Reid, G. A., and Chapman, S. K. (1994) Biochem. J. 301, 829-834], and the A198G/L230A double mutant enzyme is 6-fold more efficient with the aromatic substrate l-mandelate than it is with l-lactate [Sinclair, R., Reid, G. A., and Chapman, S. K. (1998) Biochem. J. 333, 117-120]. To complement these solution studies, we have solved the structure of the A198G/L230A enzyme in complex with pyruvate and as the FMN-sulfite adduct (both to 2.7 A resolution). We have also obtained the structure of the L230A mutant enzyme in complex with phenylglyoxylate (the product of mandelate oxidation) to 3.0 A resolution. These structures reveal the increased active-site volume available for binding larger substrates, while also confirming that the integrity of the interactions important for catalysis is maintained. In addition to this, the mode of binding of the bulky phenylglyoxylate at the active site is in accordance with the operation of a hydride transfer mechanism for substrate oxidation/flavin reduction in flavocytochrome b(2), whereas a mechanism involving the formation of a carbanion intermediate would appear to be sterically prohibited.

Articles - 1sze mentioned but not cited (2)

  1. Coupling Protein Side-Chain and Backbone Flexibility Improves the Re-design of Protein-Ligand Specificity. Ollikainen N, de Jong RM, Kortemme T. PLoS Comput Biol 11 e1004335 (2015)
  2. Comparison of Rosetta flexible-backbone computational protein design methods on binding interactions. Loshbaugh AL, Kortemme T. Proteins 88 206-226 (2020)


Reviews citing this publication (8)

  1. Oxidation of amines by flavoproteins. Fitzpatrick PF. Arch Biochem Biophys 493 13-25 (2010)
  2. Cholesterol oxidase: biochemistry and structural features. Vrielink A, Ghisla S. FEBS J 276 6826-6843 (2009)
  3. Microbial lactate utilization: enzymes, pathogenesis, and regulation. Jiang T, Gao C, Ma C, Xu P. Trends Microbiol 22 589-599 (2014)
  4. Enantiocomplementary enzymes: classification, molecular basis for their enantiopreference, and prospects for mirror-image biotransformations. Mugford PF, Wagner UG, Jiang Y, Faber K, Kazlauskas RJ. Angew Chem Int Ed Engl 47 8782-8793 (2008)
  5. Flavin-containing heme enzymes. Mowat CG, Gazur B, Campbell LP, Chapman SK. Arch Biochem Biophys 493 37-52 (2010)
  6. Restimulation-induced cell death: new medical and research perspectives. Zheng L, Li J, Lenardo M. Immunol Rev 277 44-60 (2017)
  7. Direct Electron Transfer of Enzymes Facilitated by Cytochromes. Ma S, Ludwig R. ChemElectroChem 6 958-975 (2019)
  8. Another look at the interaction between mitochondrial cytochrome c and flavocytochrome b (2). Lederer F. Eur Biophys J 40 1283-1299 (2011)

Articles citing this publication (11)

  1. Metal ions in biological catalysis: from enzyme databases to general principles. Andreini C, Bertini I, Cavallaro G, Holliday GL, Thornton JM. J Biol Inorg Chem 13 1205-1218 (2008)
  2. Crystal structure of LAAO from Calloselasma rhodostoma with an L-phenylalanine substrate: insights into structure and mechanism. Moustafa IM, Foster S, Lyubimov AY, Vrielink A. J Mol Biol 364 991-1002 (2006)
  3. Active site and loop 4 movements within human glycolate oxidase: implications for substrate specificity and drug design. Murray MS, Holmes RP, Lowther WT. Biochemistry 47 2439-2449 (2008)
  4. X-ray structures of Aerococcus viridans lactate oxidase and its complex with D-lactate at pH 4.5 show an alpha-hydroxyacid oxidation mechanism. Furuichi M, Suzuki N, Dhakshnamoorhty B, Minagawa H, Yamagishi R, Watanabe Y, Goto Y, Kaneko H, Yoshida Y, Yagi H, Waga I, Kumar PK, Mizuno H. J Mol Biol 378 436-446 (2008)
  5. Crystallographic study on the interaction of L-lactate oxidase with pyruvate at 1.9 Angstrom resolution. Li SJ, Umena Y, Yorita K, Matsuoka T, Kita A, Fukui K, Morimoto Y. Biochem Biophys Res Commun 358 1002-1007 (2007)
  6. L-lactate dehydrogenation in flavocytochrome b2: a first principles molecular dynamics study. Tabacchi G, Zucchini D, Caprini G, Gamba A, Lederer F, Vanoni MA, Fois E. FEBS J 276 2368-2380 (2009)
  7. QM/MM study of l-lactate oxidation by flavocytochrome b2. Gillet N, Ruiz-Pernía JJ, de la Lande A, Lévy B, Lederer F, Demachy I, Moliner V. Phys Chem Chem Phys 18 15609-15618 (2016)
  8. The Ala95-to-Gly substitution in Aerococcus viridans l-lactate oxidase revisited - structural consequences at the catalytic site and effect on reactivity with O2 and other electron acceptors. Stoisser T, Rainer D, Leitgeb S, Wilson DK, Nidetzky B. FEBS J 282 562-578 (2015)
  9. High-resolution structures of cholesterol oxidase in the reduced state provide insights into redox stabilization. Golden E, Karton A, Vrielink A. Acta Crystallogr D Biol Crystallogr 70 3155-3166 (2014)
  10. A double mutant of highly purified Geobacillus stearothermophilus lactate dehydrogenase recognises l-mandelic acid as a substrate. Binay B, Sessions RB, Karagüler NG. Enzyme Microb Technol 52 393-399 (2013)
  11. Further evidence in favour of a carbanion mechanism for glycolate oxidase. Pasquier H, Lederer F. FEBS Open Bio 13 938-950 (2023)


Quick links