title: Effect of metal binding and posttranslational lysine carboxylation on the activity of recombinant hydantoinase.
abstract: Bacterial hydantoinase possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carboxylated lysine. How the carboxylated lysine and metal binding affect the activity of hydantoinase was investigated. A significant amount of iron was always found in Agrobacterium radiobacter hydantoinase purified from unsupplemented cobalt-, manganese-, or zinc-amended Escherichia coli cell cultures. A titration curve for the reactivation of apohydantoinase with cobalt indicates that the first metal was preferentially bound but did not give any enzyme activity until the second metal was also attached to the hydantoinase. The pH profiles of the metal-reconstituted hydantoinase were dependent on the specific metal ion bound to the active site, indicating a direct involvement of metal in catalysis. Mutation of the metal binding site residues, H57A, H59A, K148A, H181A, H237A, and D313A, completely abolished hydantoinase activity but preserved about half of the metal content, except for K148A, which lost both metals in its active site. However, the activity of K148A could be chemically rescued by short-chain carboxylic acids in the presence of cobalt, indicating that the carboxylated lysine was needed to coordinate the binuclear ion within the active site of hydantoinase. The mutant D313E enzyme was also active but resulted in a pH profile different from that of wild-type hydantoinase. A mechanism for hydantoinase involving metal, carboxylated K148, and D313 was proposed.
authors.affiliation: Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan.
authors.forename: Cheng-Yang
authors.initials: CY
authors.lastname: Huang
authors.forename: Ching-Chen
authors.initials: CC
authors.lastname: Hsu
authors.forename: Mei-Chun
authors.initials: MC
authors.lastname: Chen
authors.forename: Yuh-Shyong
authors.initials: YS
authors.lastname: Yang
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002264:Carboxylic Acids / Q000737:chemistry; D002384:Catalysis; D003035:Cobalt / Q000378:metabolism*; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism*; D015394:Molecular Structure; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012231:Rhizobium / Q000201:enzymology*; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism*
chemical_list: D002264:Carboxylic Acids; D011994:Recombinant Proteins; D003035:Cobalt; D000581:Amidohydrolases; C020047:dihydropyrimidinase; D015032:Zinc; D008239:Lysine
references.citation: Methods Enzymol. 1979;63:103-38
references.pmid: 502857
references.citation: Biochemistry. 2000 Jun 27;39(25):7357-64
references.pmid: 10858282
references.citation: Bacteriol Rev. 1976 Jun;40(2):403-68
references.pmid: 786256
references.citation: Biochemistry. 2005 May 3;44(17):6383-91
references.pmid: 15850372
references.citation: J Biol Chem. 1946 Jun;163:683-5
references.pmid: 20985639
references.citation: Nucleic Acids Res. 2003 Mar 15;31(6):1683-92
references.pmid: 12626710
references.citation: Nat Rev Mol Cell Biol. 2003 Feb;4(2):105-16
references.pmid: 12563288
references.citation: Nat Biotechnol. 2000 Mar;18(3):317-20
references.pmid: 10700149
references.citation: Biochemistry. 2004 May 18;43(19):5707-15
references.pmid: 15134445
references.citation: J Mol Biol. 2005 Mar 18;347(1):95-105
references.pmid: 15733920
references.citation: Biochemistry. 2002 Jul 30;41(30):9410-7
references.pmid: 12135362
references.citation: Biochemistry. 1997 Feb 25;36(8):1982-8
references.pmid: 9047295
references.citation: J Mol Biol. 2003 Sep 5;332(1):243-56
references.pmid: 12946361
references.citation: J Biol Chem. 1949 Dec;181(2):449-58
references.pmid: 15393763
references.citation: Annu Rev Biochem. 1999;68:33-57
references.pmid: 10872443
references.citation: J Biol Chem. 1993 May 25;268(15):10870-5
references.pmid: 8388376
references.citation: Biochemistry. 1991 May 28;30(21):5172-7
references.pmid: 1903652
references.citation: J Mol Biol. 2002 Jun 28;320(1):143-56
references.pmid: 12079340
references.citation: Nat Struct Biol. 1998 Dec;5(12):1053-8
references.pmid: 9846875
references.citation: Science. 2003 Mar 14;299(5613):1694-7
references.pmid: 12637735
references.citation: Biochemistry. 1999 Aug 3;38(31):9964-70
references.pmid: 10433703
references.citation: Nat Struct Biol. 2003 Apr;10(4):234-6
references.pmid: 12660715
references.citation: Biochemistry. 1995 Jun 27;34(25):7973-8
references.pmid: 7794910
references.citation: Biochemistry. 2001 Jun 19;40(24):6989-97
references.pmid: 11401542
references.citation: Biochem Biophys Res Commun. 2002 Oct 4;297(4):1027-32
references.pmid: 12359259
references.citation: Proteins. 1997 May;28(1):72-82
references.pmid: 9144792
references.citation: Science. 1995 May 19;268(5213):998-1004
references.pmid: 7754395
references.citation: Annu Rev Biochem. 2001;70:209-46
references.pmid: 11395407
references.citation: J Biol Chem. 1957 May;226(1):277-88
references.pmid: 13428761
references.citation: Biol Chem. 1998 Jun;379(6):743-7
references.pmid: 9687026
references.citation: J Biol Inorg Chem. 2006 Apr;11(3):351-8
references.pmid: 16489411
references.citation: J Biol Inorg Chem. 2002 Sep;7(7-8):704-12
references.pmid: 12203007
references.citation: J Mol Biol. 2002 Jan 25;315(4):687-97
references.pmid: 11812140
references.citation: Biochemistry. 1999 Aug 24;38(34):11079-85
references.pmid: 10460163
references.citation: J Biol Inorg Chem. 2006 Jun;11(4):398-408
references.pmid: 16596389
references.citation: Chem Rev. 2004 Feb;104(2):509-25
references.pmid: 14871133
references.citation: Biochemistry. 2004 Dec 28;43(51):16285-92
references.pmid: 15610022
references.citation: Protein Expr Purif. 2003 Jul;30(1):134-9
references.pmid: 12821331
references.citation: Biochem Biophys Res Commun. 2003 Dec 12;312(2):467-72
references.pmid: 14637160
references.citation: Curr Opin Biotechnol. 2001 Dec;12(6):559-63
references.pmid: 11849938
references.citation: Curr Opin Chem Biol. 2003 Apr;7(2):252-64
references.pmid: 12714059
references.citation: J Biol Inorg Chem. 2004 Jun;9(4):429-38
references.pmid: 15067523
references.citation: J Biol Chem. 1984 Mar 10;259(5):3293-8
references.pmid: 6142052
references.citation: Biochem J. 1998 Feb 15;330 ( Pt 1):295-302
references.pmid: 9537960
references.citation: Appl Microbiol Biotechnol. 1999 Mar;51(3):293-309
references.pmid: 10222578
references.citation: J Bacteriol. 2003 Jul;185(14):4038-49
references.pmid: 12837777