Mutational and Structural Analysis of L-N-carbamoylase Reveals New Insights into a Peptidase M20/M25/M40 Family Member

Overview

Journal J Bacteriol

Publisher American Society for Microbiology

Specialty Microbiology

Date 2012 Aug 21

PMID 22904279

Citations 6

Authors

Sergio Martinez-Rodriguez

Abel Garcia-Pino

Francisco Javier Las Heras-Vazquez

Josefa Maria Clemente-Jimenez

Felipe Rodriguez-Vico

Juan M Garcia-Ruiz

Remy Loris

Jose Antonio Gavira

Affiliations

Soon will be listed here.

Abstract

N-Carbamoyl-L-amino acid amidohydrolases (L-carbamoylases) are important industrial enzymes used in kinetic resolution of racemic mixtures of N-carbamoyl-amino acids due to their strict enantiospecificity. In this work, we report the first L-carbamoylase structure belonging to Geobacillus stearothermophilus CECT43 (BsLcar), at a resolution of 2.7 Å. Structural analysis of BsLcar and several members of the peptidase M20/M25/M40 family confirmed the expected conserved residues at the active site in this family, and site-directed mutagenesis revealed their relevance to substrate binding. We also found an unexpectedly conserved arginine residue (Arg(234) in BsLcar), proven to be critical for dimerization of the enzyme. The mutation of this sole residue resulted in a total loss of activity and prevented the formation of the dimer in BsLcar. Comparative studies revealed that the dimerization domain of the peptidase M20/M25/M40 family is a "small-molecule binding domain," allowing further evolutionary considerations for this enzyme family.

Citing Articles

Exploring the Kinetics and Thermodynamics of a Novel Histidine Ammonia-Lyase from .

Salas-Garrucho F, Carrillo-Moreno A, Contreras L, Rodriguez-Vico F, Clemente-Jimenez J, Las Heras-Vazquez F Int J Mol Sci. 2024; 25(18).

PMID: 39337646 PMC: 11432326. DOI: 10.3390/ijms251810163.

Structural and biochemical characterisation of the N-carbamoyl-β-alanine amidohydrolase from Rhizobium radiobacter MDC 8606.

Paloyan A, Sargsyan A, Karapetyan M, Hambardzumyan A, Kocharov S, Panosyan H FEBS J. 2023; 290(23):5566-5580.

PMID: 37634202 PMC: 10952681. DOI: 10.1111/febs.16943.

Open Issues for Protein Function Assignment in and Other Halophilic Archaea.

Pfeiffer F, Dyall-Smith M Genes (Basel). 2021; 12(7).

PMID: 34202810 PMC: 8305020. DOI: 10.3390/genes12070963.

A fundamental catalytic difference between zinc and manganese dependent enzymes revealed in a bacterial isatin hydrolase.

Sommer T, Bjerregaard-Andersen K, Uribe L, Etzerodt M, Diezemann G, Gauss J Sci Rep. 2018; 8(1):13104.

PMID: 30166577 PMC: 6117287. DOI: 10.1038/s41598-018-31259-y.

Enzymatic hydrolysis by transition-metal-dependent nucleophilic aromatic substitution.

Kalyoncu S, Heaner Jr D, Kurt Z, Bethel C, Ukachukwu C, Chakravarthy S Nat Chem Biol. 2016; 12(12):1031-1036.

PMID: 27694799 PMC: 5110390. DOI: 10.1038/nchembio.2191.

References

Holm L, Rosenstrom P . Dali server: conservation mapping in 3D. Nucleic Acids Res. 2010; 38(Web Server issue):W545-9. PMC: 2896194. DOI: 10.1093/nar/gkq366. View

Agarwal R, Burley S, Swaminathan S . Structural analysis of a ternary complex of allantoate amidohydrolase from Escherichia coli reveals its mechanics. J Mol Biol. 2007; 368(2):450-63. DOI: 10.1016/j.jmb.2007.02.028. View

Davis I, Leaver-Fay A, Chen V, Block J, Kapral G, Wang X . MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 2007; 35(Web Server issue):W375-83. PMC: 1933162. DOI: 10.1093/nar/gkm216. View

Corazza A, Rosano C, Pagano K, Alverdi V, Esposito G, Capanni C . Structure, conformational stability, and enzymatic properties of acylphosphatase from the hyperthermophile Sulfolobus solfataricus. Proteins. 2005; 62(1):64-79. DOI: 10.1002/prot.20703. View

Aravind L, Koonin E . Gleaning non-trivial structural, functional and evolutionary information about proteins by iterative database searches. J Mol Biol. 1999; 287(5):1023-40. DOI: 10.1006/jmbi.1999.2653. View

Laskowski R, Chistyakov V, Thornton J . PDBsum more: new summaries and analyses of the known 3D structures of proteins and nucleic acids. Nucleic Acids Res. 2004; 33(Database issue):D266-8. PMC: 539955. DOI: 10.1093/nar/gki001. View

Kerfeld C, Sawaya M, Tanaka S, Nguyen C, Phillips M, Beeby M . Protein structures forming the shell of primitive bacterial organelles. Science. 2005; 309(5736):936-8. DOI: 10.1126/science.1113397. View

Lundgren S, Gojkovic Z, Piskur J, Dobritzsch D . Yeast beta-alanine synthase shares a structural scaffold and origin with dizinc-dependent exopeptidases. J Biol Chem. 2003; 278(51):51851-62. DOI: 10.1074/jbc.M308674200. View

Girish T, Gopal B . Crystal structure of Staphylococcus aureus metallopeptidase (Sapep) reveals large domain motions between the manganese-bound and apo-states. J Biol Chem. 2010; 285(38):29406-15. PMC: 2937973. DOI: 10.1074/jbc.M110.147579. View

10.

Hutchinson E, Thornton J . PROMOTIF--a program to identify and analyze structural motifs in proteins. Protein Sci. 1996; 5(2):212-20. PMC: 2143354. DOI: 10.1002/pro.5560050204. View

11.

Martinez-Gomez A, Martinez-Rodriguez S, Pozo-Dengra J, Tessaro D, Servi S, Clemente-Jimenez J . Potential application of N-carbamoyl-beta-alanine amidohydrolase from Agrobacterium tumefaciens C58 for beta-amino acid production. Appl Environ Microbiol. 2008; 75(2):514-20. PMC: 2620706. DOI: 10.1128/AEM.01128-08. View

12.

Ettema T, Brinkman A, Tani T, Rafferty J, van der Oost J . A novel ligand-binding domain involved in regulation of amino acid metabolism in prokaryotes. J Biol Chem. 2002; 277(40):37464-8. DOI: 10.1074/jbc.M206063200. View

13.

Murshudov G, Skubak P, Lebedev A, Pannu N, Steiner R, Nicholls R . REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr. 2011; 67(Pt 4):355-67. PMC: 3069751. DOI: 10.1107/S0907444911001314. View

14.

Lindner H, Lunin V, Alary A, Hecker R, Cygler M, Menard R . Essential roles of zinc ligation and enzyme dimerization for catalysis in the aminoacylase-1/M20 family. J Biol Chem. 2003; 278(45):44496-504. DOI: 10.1074/jbc.M304233200. View

15.

Hu H, Hsu W, Chien H . Characterization and phylogenetic analysis of a thermostable N-carbamoyl- l-amino acid amidohydrolase from Bacillus kaustophilus CCRC11223. Arch Microbiol. 2003; 179(4):250-7. DOI: 10.1007/s00203-003-0524-9. View

16.

Guex N, Peitsch M . SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis. 1998; 18(15):2714-23. DOI: 10.1002/elps.1150181505. View

17.

Holz R, Bzymek K, Swierczek S . Co-catalytic metallopeptidases as pharmaceutical targets. Curr Opin Chem Biol. 2003; 7(2):197-206. DOI: 10.1016/s1367-5931(03)00033-4. View

18.

Martinez-Rodriguez S, Martinez-Gomez A, Rodriguez-Vico F, Clemente-Jimenez J, Las Heras-Vazquez F . Carbamoylases: characteristics and applications in biotechnological processes. Appl Microbiol Biotechnol. 2009; 85(3):441-58. DOI: 10.1007/s00253-009-2250-y. View

19.

Ogawa J, Shimizu S . Beta-ureidopropionase with N-carbamoyl-alpha-L-amino acid amidohydrolase activity from an aerobic bacterium, Pseudomonas putida IFO 12996. Eur J Biochem. 1994; 223(2):625-30. DOI: 10.1111/j.1432-1033.1994.tb19034.x. View

20.

Dey S, Grant G, Sacchettini J . Crystal structure of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase: extreme asymmetry in a tetramer of identical subunits. J Biol Chem. 2005; 280(15):14892-9. DOI: 10.1074/jbc.M414489200. View