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Chemical rescue of the post-translationally carboxylated lysine mutant of allantoinase and dihydroorotase by metal ions and short-chain carboxylic acids

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Abstract

Bacterial allantoinase (ALLase) and dihydroorotase (DHOase) are members of the cyclic amidohydrolase family. ALLase and DHOase possess similar binuclear metal centers in the active site in which two metals are bridged by a post-translationally carboxylated lysine. In this study, we determined the effects of carboxylated lysine and metal binding on the activities of ALLase and DHOase. Although DHOase is a metalloenzyme, purified DHOase showed high activity without additional metal supplementation in a reaction mixture or bacterial culture. However, unlike DHOase, ALLase had no activity unless some specific metal ions were added to the reaction mixture or culture. Substituting the metal binding sites H59, H61, K146, H186, H242, or D315 with alanine completely abolished the activity of ALLase. However, the K146C, K146D and K146E mutants of ALLase were still active with about 1–6 % activity of the wild-type enzyme. These ALLase K146 mutants were found to have 1.4–1.7 mol metal per mole enzyme subunit, which may indicate that they still contained the binuclear metal center in the active site. The activity of the K146A mutant of the ALLase and the K103A mutant of DHOase can be chemically rescued by short-chain carboxylic acids, such as acetic, propionic, and butyric acids, but not by ethanol, propan-1-ol, and imidazole, in the presence of Co2+ or Mn2+ ions. However, the activity was still ~10-fold less than that of wild-type ALLase. Overall, these results indicated that the 20 natural basic amino acid residues were not sufficiently able to play the role of lysine. Accordingly, we proposed that during evolution, the post-translational modification of carboxylated lysine in the cyclic amidohydrolase family was selected for promoting binuclear metal center self-assembly and increasing the nucleophilicity of the hydroxide at the active site for enzyme catalysis. This kind of chemical rescue combined with site-directed mutagenesis may also be used to identify a binuclear metal center in the active site for other metalloenzymes.

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Abbreviations

ALLase:

Allantoinase

DHOase:

Dihydroorotase

SDS-PAGE:

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

8-HQSA:

8-Hydroxy-5-quinolinesulfonic acid

EDTA:

Ethylenediamine tetraacetic acid

IPTG:

Isopropyl thiogalactoside

ICP-MS:

Inductively coupled plasma mass spectrometry

References

  • Abendroth J, Niefind K, Schomburg D (2002) X-ray structure of a dihydropyrimidinase from Thermus sp. at 1.3 Å resolution. J Mol Biol 320(1):143–156

    Article  PubMed  CAS  Google Scholar 

  • Altenbuchner J, Siemann-Herzberg M, Syldatk C (2001) Hydantoinases and related enzymes as biocatalysts for the synthesis of unnatural chiral amino acids. Curr Opin Biotechnol 12(6):559–563

    Article  PubMed  CAS  Google Scholar 

  • Brown DC, Collins KD (1991) Dihydroorotase from Escherichia coli. Substitution of Co(II) for the active site Zn(II). J Biol Chem 266(3):1597–1604

    PubMed  CAS  Google Scholar 

  • Cheon YH, Kim HS, Han KH, Abendroth J, Niefind K, Schomburg D, Wang J, Kim Y (2002) Crystal structure of D-hydantoinase from Bacillus stearothermophilus: insight into the stereochemistry of enantioselectivity. Biochemistry 41(30):9410–9417

    Article  PubMed  CAS  Google Scholar 

  • Christopherson RI, Jones ME (1980) The overall synthesis of L-5,6-dihydroorotate by multienzymatic protein pyr1-3 from hamster cells. Kinetic studies, substrate channeling, and the effects of inhibitors. J Biol Chem 255(23):11381–11395

    PubMed  CAS  Google Scholar 

  • Evans DR, Guy HI (2004) Mammalian pyrimidine biosynthesis: fresh insights into an ancient pathway. J Biol Chem 279(32):33035–33038

    Article  PubMed  CAS  Google Scholar 

  • Gerlt JA, Babbitt PC (2001) Divergent evolution of enzymatic function: mechanistically diverse superfamilies and functionally distinct suprafamilies. Annu Rev Biochem 70:209–246

    Article  PubMed  CAS  Google Scholar 

  • Gerlt JA, Raushel FM (2003) Evolution of function in (beta/alpha)8-barrel enzymes. Curr Opin Chem Biol 7(2):252–264

    Article  PubMed  CAS  Google Scholar 

  • Gerlt JA, Allen KN, Almo SC, Armstrong RN, Babbitt PC, Cronan JE, Dunaway-Mariano D, Imker HJ, Jacobson MP, Minor W, Poulter CD, Raushel FM, Sali A, Shoichet BK, Sweedler JV (2011) The enzyme function initiative. Biochemistry 50(46):9950–9962

    Article  PubMed  CAS  Google Scholar 

  • Hayashi S, Fujiwara S, Noguchi T (2000) Evolution of urate-degrading enzymes in animal peroxisomes. Cell Biochem Biophys 32:123–129

    Article  PubMed  CAS  Google Scholar 

  • Ho YY, Hsieh HC, Huang CY (2011) Biochemical characterization of allantoinase from Escherichia coli BL21. Protein J 30(6):384–394

    Article  PubMed  CAS  Google Scholar 

  • Holm L, Sander C (1997) An evolutionary treasure: unification of a broad set of amidohydrolases related to urease. Proteins 28(1):72–82

    Article  PubMed  CAS  Google Scholar 

  • Hsieh HC, Huang CY (2011) Identification of a novel protein, PriB, in Klebsiella pneumoniae. Biochem Biophys Res Commun 404(1):546–551

    Article  PubMed  CAS  Google Scholar 

  • Huang YH, Huang CY (2012) Characterization of a single-stranded DNA-binding protein from Klebsiella pneumoniae: mutation at either Arg73 or Ser76 causes a less cooperative complex on DNA. Genes Cells 17:146–157

    Article  PubMed  CAS  Google Scholar 

  • Huang CY, Yang YS (2002) The role of metal on imide hydrolysis: metal content and pH profiles of metal ion-replaced mammalian imidase. Biochem Biophys Res Commun 297(4):1027–1032

    Article  PubMed  CAS  Google Scholar 

  • Huang CY, Yang YS (2003) A novel cold-adapted imidase from fish Oreochromis niloticus that catalyzes hydrolysis of maleimide. Biochem Biophys Res Commun 312(2):467–472

    Article  PubMed  CAS  Google Scholar 

  • Huang DT, Thomas MA, Christopherson RI (1999) Divalent metal derivatives of the hamster dihydroorotase domain. Biochemistry 38(31):9964–9970

    Article  PubMed  CAS  Google Scholar 

  • Huang CY, Hsu CH, Sun YJ, Wu HN, Hsiao CD (2006) Complexed crystal structure of replication restart primosome protein PriB reveals a novel single-stranded DNA-binding mode. Nucleic Acids Res 34(14):3878–3886

    Article  PubMed  CAS  Google Scholar 

  • Huang CY, Hsu CC, Chen MC, Yang YS (2009) Effect of metal binding and posttranslational lysine carboxylation on the activity of recombinant hydantoinase. J Biol Inorg Chem 14(1):111–121

    Article  PubMed  CAS  Google Scholar 

  • Huang YH, Lo YH, Huang W, Huang CY (2012) Crystal structure and DNA-binding mode of Klebsiella pneumoniae primosomal PriB protein. Genes Cells 17:837–849

    Article  PubMed  CAS  Google Scholar 

  • Kim GJ, Kim HS (1998) Identification of the structural similarity in the functionally related amidohydrolases acting on the cyclic amide ring. Biochem J 330:295–302

    PubMed  CAS  Google Scholar 

  • Kim GJ, Lee DE, Kim HS (2000) Functional expression and characterization of the two cyclic amidohydrolase enzymes, allantoinase and a novel phenylhydantoinase, from Escherichia coli. J Bacteriol 182(24):7021–7028

    Article  PubMed  CAS  Google Scholar 

  • Kim K, Kim MI, Chung J, Ahn JH, Rhee S (2009) Crystal structure of metal-dependent allantoinase from Escherichia coli. J Mol Biol 387(5):1067–1074

    Article  PubMed  CAS  Google Scholar 

  • Kumar V, Saxena N, Sarma M, Radha Kishan KV (2011) Carboxylated lysine is required for higher activities in Hydantoinases. Protein Pept Lett 18(7):663–669

    Article  PubMed  CAS  Google Scholar 

  • Kuo JM, Chae MY, Raushel FM (1997) Perturbations to the active site of phosphotriesterase. Biochemistry 36(8):1982–1988

    Article  PubMed  CAS  Google Scholar 

  • Landau M, Mayrose I, Rosenberg Y, Glaser F, Martz E, Pupko T, Ben-Tal N (2005) ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res 33:W299–W302

    Article  PubMed  CAS  Google Scholar 

  • Li J, Cross JB, Vreven T, Meroueh SO, Mobashery S, Schlegel HB (2005) Lysine carboxylation in proteins: OXA-10 beta-lactamase. Proteins 61(2):246–257

    Article  PubMed  CAS  Google Scholar 

  • Lohkamp B, Andersen B, Piskur J, Dobritzsch D (2006) The crystal structures of dihydropyrimidinases reaffirm the close relationship between cyclic amidohydrolases and explain their substrate specificity. J Biol Chem 281(19):13762–13776

    Article  PubMed  CAS  Google Scholar 

  • Mally MI, Grayson DR, Evans DR (1980) Catalytic synergy in the multifunctional protein that initiates pyrimidine biosynthesis in Syrian hamster cells. J Biol Chem 255(23):11372–11380

    PubMed  CAS  Google Scholar 

  • Martin PD, Purcarea C, Zhang P, Vaishnav A, Sadecki S, Guy-Evans HI, Evans DR, Edwards BF (2005) The crystal structure of a novel, latent dihydroorotase from Aquifex aeolicus at 1.7 Å resolution. J Mol Biol 348(3):535–547

    Article  PubMed  CAS  Google Scholar 

  • Martinez-Rodriguez S, Martinez-Gomez AI, Clemente-Jimenez JM, Rodriguez-Vico F, Garcia-Ruiz JM, Las Heras-Vazquez FJ, Gavira JA (2010a) Structure of dihydropyrimidinase from Sinorhizobium meliloti CECT4114: new features in an amidohydrolase family member. J Struct Biol 169(2):200–208

    Article  PubMed  CAS  Google Scholar 

  • Martinez-Rodriguez S, Martinez-Gomez AI, Rodriguez-Vico F, Clemente-Jimenez JM, Las Heras-Vazquez FJ (2010b) Carbamoylases: characteristics and applications in biotechnological processes. Appl Microbiol Biotechnol 85(3):441–458

    Article  PubMed  CAS  Google Scholar 

  • McClelland M, Sanderson KE, Spieth J, Clifton SW, Latreille P, Courtney L, Porwollik S, Ali J, Dante M, Du F, Hou S, Layman D, Leonard S, Nguyen C, Scott K, Holmes A, Grewal N, Mulvaney E, Ryan E, Sun H, Florea L, Miller W, Stoneking T, Nhan M, Waterston R, Wilson RK (2001) Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413(6858):852–856

    Article  PubMed  CAS  Google Scholar 

  • McPhail D, Shepherdson M (2006) The aspartate transcarbamoylase-dihydroorotase complex in Deinococcus radiophilus has an active dihydroorotase. Arch Microbiol 185(1):78–81

    Article  PubMed  CAS  Google Scholar 

  • Mehboob S, Mulhearn DC, Truong K, Johnson ME, Santarsiero BD (2010) Structure of dihydroorotase from Bacillus anthracis at 2.6 Å resolution. Acta Crystallogr, Sect F: Struct Biol Cryst Commun 66(11):1432–1435

    Article  Google Scholar 

  • Mulrooney SB, Hausinger RP (2003) Metal ion dependence of recombinant Escherichia coli allantoinase. J Bacteriol 185(1):126–134

    Article  PubMed  CAS  Google Scholar 

  • Nara T, Hashimoto M, Hirawake H, Liao CW, Fukai Y, Suzuki S, Tsubouchi A, Morales J, Takamiya S, Fujimura T, Taka H, Mineki R, Fan CK, Inaoka DK, Inoue M, Tanaka A, Harada S, Kita K, Aoki T (2012) Molecular interaction of the first 3 enzymes of the de novo pyrimidine biosynthetic pathway of Trypanosoma cruzi. Biochem Biophys Res Commun 418(1):140–143

    Article  PubMed  CAS  Google Scholar 

  • Park IS, Hausinger RP (1995) Requirement of carbon dioxide for in vitro assembly of the urease nickel metallocenter. Science 267(5201):1156–1158

    Article  PubMed  CAS  Google Scholar 

  • Pearson MA, Schaller RA, Michel LO, Karplus PA, Hausinger RP (1998) Chemical rescue of Klebsiella aerogenes urease variants lacking the carbamylated-lysine nickel ligand. Biochemistry 37(17):6214–6220

    Article  PubMed  CAS  Google Scholar 

  • Porter TN, Li Y, Raushel FM (2004) Mechanism of the dihydroorotase reaction. Biochemistry 43(51):16285–16292

    Article  PubMed  CAS  Google Scholar 

  • Radha Kishan KV, Vohra RM, Ganesan K, Agrawal V, Sharma VM, Sharma R (2005) Molecular structure of D-hydantoinase from Bacillus sp. AR9: evidence for mercury inhibition. J Mol Biol 347(1):95–105

    Article  PubMed  CAS  Google Scholar 

  • Ramazzina I, Cendron L, Folli C, Berni R, Monteverdi D, Zanotti G, Percudani R (2008) Logical identification of an allantoinase analog (puuE) recruited from polysaccharide deacetylases. J Biol Chem 283(34):23295–23304

    Article  PubMed  CAS  Google Scholar 

  • Schneider KD, Karpen ME, Bonomo RA, Leonard DA, Powers RA (2009) The 1.4 A crystal structure of the class D beta-lactamase OXA-1 complexed with doripenem. Biochemistry 48(50):11840–11847

    Article  PubMed  CAS  Google Scholar 

  • Seibert CM, Raushel FM (2005) Structural and catalytic diversity within the amidohydrolase superfamily. Biochemistry 44(17):6383–6391

    Article  PubMed  CAS  Google Scholar 

  • Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150(1):76–85

    Article  PubMed  CAS  Google Scholar 

  • Thoden JB, Phillips GN Jr, Neal TM, Raushel FM, Holden HM (2001) Molecular structure of dihydroorotase: a paradigm for catalysis through the use of a binuclear metal center. Biochemistry 40(24):6989–6997

    Article  PubMed  CAS  Google Scholar 

  • Wang CC, Tsau HW, Chen WT, Huang CY (2010) Identification and characterization of a putative dihydroorotase, KPN01074, from Klebsiella pneumoniae. Protein J 29(6):445–452

    Article  PubMed  CAS  Google Scholar 

  • Washabaugh MW, Collins KD (1984) Dihydroorotase from Escherichia coli. Purification and characterization. J Biol Chem 259(5):3293–3298

    PubMed  CAS  Google Scholar 

  • Washabaugh MW, Collins KD (1986) Dihydroorotase from Escherichia coli. Sulfhydryl group-metal ion interactions. J Biol Chem 261(13):5920–5929

    PubMed  CAS  Google Scholar 

  • Xu Z, Liu Y, Yang Y, Jiang W, Arnold E, Ding J (2003) Crystal structure of D-Hydantoinase from Burkholderia pickettii at a resolution of 2.7 Angstroms: insights into the molecular basis of enzyme thermostability. J Bacteriol 185(14):4038–4049

    Article  PubMed  CAS  Google Scholar 

  • Zhang P, Martin PD, Purcarea C, Vaishnav A, Brunzelle JS, Fernando R, Guy-Evans HI, Evans DR, Edwards BF (2009) Dihydroorotase from the hyperthermophile Aquifex aeolicus is activated by stoichiometric association with aspartate transcarbamoylase and forms a one-pot reactor for pyrimidine biosynthesis. Biochemistry 48(4):766–778

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We would like to thank three anonymous reviewers and the editor for their comments. This research was supported by a grant from the National Research Program for Genome Medicine, Taiwan (NSC 100-3112-B-040-001 to C.Y. Huang).

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None declared.

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Authors and Affiliations

  1. Department of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Chien-Kuo N. Rd., Taichung, Taiwan

    Ya-Yeh Ho, Yen-Hua Huang & Cheng-Yang Huang

  2. Department of Medical Research, Chung Shan Medical University Hospital, No.110, Sec.1, Chien-Kuo N. Rd., Taichung, Taiwan

    Cheng-Yang Huang

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  1. Ya-Yeh Ho
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Correspondence to Cheng-Yang Huang.

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Ho, YY., Huang, YH. & Huang, CY. Chemical rescue of the post-translationally carboxylated lysine mutant of allantoinase and dihydroorotase by metal ions and short-chain carboxylic acids. Amino Acids 44, 1181–1191 (2013). https://doi.org/10.1007/s00726-012-1451-3

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