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Crystal structures and biochemical analyses of the bacterial arginine dihydrolase ArgZ suggests a "bond rotation" catalytic mechanism.

ABSTRACT: A recently discovered ornithine-ammonia cycle (OAC) serves as a conduit in the nitrogen storage and remobilization machinery in cyanobacteria. The OAC involves an arginine catabolic reaction catalyzed by the arginine dihydrolase ArgZ whose catalytic mechanism is unknown. Here we determined the crystal structures at 1.2-3.0 Å of unliganded ArgZ from the cyanobacterium Synechocystis sp. PCC6803 and of ArgZ complexed with its substrate arginine, a covalently linked reaction intermediate, or the reaction product ornithine. The structures reveal that a key residue, Asn⁷¹, in the ArgZ active center functions as the determinant distinguishing ArgZ from other members of the guanidino group-modifying enzyme superfamily. The structures, along with biochemical evidence from enzymatic assays coupled with electrospray ionization MS techniques, further suggest that ArgZ-catalyzed conversion of arginine to ornithine, ammonia, and carbon dioxide consists of two successive cycles of amine hydrolysis. Finally, we show that arginine dihydrolases are broadly distributed among bacteria and metazoans, suggesting that the OAC may be frequently used for redistribution of nitrogen from arginine catabolism or nitrogen fixation.

SUBMITTER: Zhuang N

PROVIDER: S-EPMC7029115 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Crystal structures and biochemical analyses of the bacterial arginine dihydrolase ArgZ suggests a "bond rotation" catalytic mechanism.

Zhuang Ningning N Zhang Hao H Li Lingting L Wu Xiaoxian X Yang Chen C Zhang Yu Y

The Journal of biological chemistry 20191230 7

A recently discovered ornithine-ammonia cycle (OAC) serves as a conduit in the nitrogen storage and remobilization machinery in cyanobacteria. The OAC involves an arginine catabolic reaction catalyzed by the arginine dihydrolase ArgZ whose catalytic mechanism is unknown. Here we determined the crystal structures at 1.2-3.0 Å of unliganded ArgZ from the cyanobacterium <i>Synechocystis</i> sp. PCC6803 and of ArgZ complexed with its substrate arginine, a covalently linked reaction intermediate, or ...[more]

PMID: 31914412

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Project description:Extracellular proteases of Porphyromonas gingivalis specific for arginyl peptide bonds are considered to be important virulence factors in periodontal disease. In order to determine the number, inter-relationship and kinetic properties of these proteases, extracellular enzymes with this peptide-bond specificity were purified and characterized from P. gingivalis W50. Three forms, which we denote RI, RI-A and RI-B, accounted for all of the activity in the supernatant. All three enzymes contain an alpha chain of approximately 54 kDa with the same N-terminal amino acid sequence. RI is a heterodimer of non-covalently linked alpha and beta chains which migrate to the same position on SDS/PAGE but which can be resolved by 8 M urea/PAGE. RI-A and RI-B are both monomeric, but the molecular mass of RI-B (70-80 kDa) is significantly increased due to post-translational modification with lipopolysaccharide. All forms show absolute specificity for peptide bonds with Arg in the P1 position and are also capable of hydrolysing N-terminal Arg and C-terminal Arg-Arg peptide bonds. Thus they show limited amino- and carboxy-peptidase activity. For the hydrolysis of Nalpha-benzoyl-L-Arg-p-nitroanilide, the pH optimum is 8.0 at 30 degrees C. The Vmax for all three enzymes is controlled by ionization of two residues with apparent pKas at 30 degrees C of 6. 5+/-0.05 and 9.7+/-0.05, and DeltaH values of approximately 29 kJ/mol and approximately 24 kJ/mol in the enzyme-substrate complex. By analogy with papain, the pKa of 6.5 could be ascribed to a Cys and the pKa of 9.7 to a His residue. E-64 [L-trans-epoxysuccinyl-leucylamide-4-(4-guanidino)butane] is a competitive inhibitor of RI, RI-A and RI-B. Based on physical properties and kinetic behaviour, RI-A appears to be analogous to gingipain from P. gingivalis HG66. However the alpha/beta structure of RI differs significantly from that of the high-molecular-mass multimeric complex of gingipain containing four haemagglutinins described by others. Since the genes for RI and high-molecular-mass gingipain are identical, the data indicate that an alternative processing pathway is involved in the formation of RI from the initial precursor. Furthermore, the identical N-termini and enzymic properties of the catalytic component of RI, RI-A and RI-B suggest that the maturation pathway of the RI precursor may also give rise to RI-A and RI-B. The physiological functions of these isoforms and their role in the disease process may become more apparent through examination of their interactions with host proteins.

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Crystal structures and biochemical analyses of the bacterial arginine dihydrolase ArgZ suggests a "bond rotation" catalytic mechanism.

Publications

Crystal structures and biochemical analyses of the bacterial arginine dihydrolase ArgZ suggests a "bond rotation" catalytic mechanism.

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