P83772 (CRNA_PSEPU) Reviewed, UniProtKB/Swiss-Prot

Names and origin

Protein names

Recommended name:
Creatinine amidohydrolase
EC=3.5.2.10

Alternative name(s):
Creatininase

Gene names

Name:

crnA

Organism

Pseudomonas putida (Arthrobacter siderocapsulatus)

Taxonomic identifier

303 [NCBI]

Taxonomic lineage

cellular organisms › Bacteria › Proteobacteria › Gammaproteobacteria › Pseudomonadales › Pseudomonadaceae › Pseudomonas › Pseudomonas putida group

Protein attributes

Sequence length	260 AA.
Sequence status	Complete.
Sequence processing	The displayed sequence is further processed into a mature form.
Protein existence	Evidence at protein level

General annotation (Comments)

Function	Cyclic amidohydrolase that catalyzes the reversible conversion of creatinine to creatine. Is also active toward glycocyamidine, though the reaction rate is very low, but it is completely inert toward hydantoin and its derivatives. Ref.2
Catalytic activity	Creatinine + H2O = creatine. Ref.2 Ref.5
Cofactor	Binds 2 Zn2+ ions per subunit. The Zn2+ in the metal 1 binding site can be replaced with Mn2+; however, the second zinc in metal binding site 2 is much more tightly bound and can not be replaced. The enzyme with one zinc and one manganese ion is more active than that with two zinc ions. Ref.3 Ref.4
Enzyme regulation	Is markedly inactivated in vitro by heavy metal ions, N-bromosuccinimide, ethoxyformic anhydride, and dye-sensitized photooxidation. Ref.2
Pathway	Amine and polyamine degradation; creatinine degradation.
Subunit structure	Homohexamer; trimer of dimers. Ref.3 Ref.4
Miscellaneous	The proposed catalytic mechanism involves two water molecules. The first molecule is a hydroxide ion that is bound as a bridge between the two metal ions and attacks the carbonyl carbon of the substrate. The second water molecule, that is bound to the carboxyl group of Glu-122 and to the metal 1, functions as a proton donor in catalysis.
Sequence similarities	Belongs to the creatininase superfamily.
Biophysicochemical properties	Kinetic parameters: KM=26 mM for creatinine Ref.2 Ref.5 KM=130 mM for creatine KM=200 mM for glycocyamidine Vmax=390 µmol/min/mg enzyme for the forward reaction (creatine formation) Vmax=1510 µmol/min/mg enzyme for the reverse reaction (creatinine formation) Vmax=3.7 µmol/min/mg enzyme with glycocyamidine as substrate pH dependence: Optimum pH is 7-9 for the forward and reverse reactions. Temperature dependence: Retains 75% of the activity after incubation at 75 degrees Celsius for 30 minutes.

Ontologies

Keywords
Ligand	Manganese Metal-binding Zinc
Molecular function	Hydrolase
Technical term	3D-structure Direct protein sequencing
Gene Ontology (GO)
Biological_process	creatine biosynthetic process Inferred from direct assay Ref.5. Source: UniProtKB creatinine catabolic process Inferred from direct assay Ref.5. Source: UniProtKB
Molecular_function	creatininase activity Inferred from direct assay Ref.5. Source: UniProtKB manganese ion binding Inferred from direct assay Ref.5. Source: UniProtKB zinc ion binding Inferred from direct assay Ref.5. Source: UniProtKB
Complete GO annotation...

Sequence annotation (Features)

Feature key

Position(s)

Length

Description

Graphical view

Feature identifier

Molecule processing

Initiator methionine

1

1

Removed Probable

Chain

2 – 260

259

Creatinine amidohydrolase

PRO_0000406934

Regions

Region

174 – 178

5

Substrate binding

Sites

Metal binding

34

1

Zinc or manganese 1

Metal binding

36

1

Zinc 2; via tele nitrogen

Metal binding

45

1

Zinc 2

Metal binding

45

1

Zinc or manganese 1

Metal binding

120

1

Zinc or manganese 1; via pros nitrogen

Metal binding

183

1

Zinc 2

Binding site

78

1

Substrate; via carbonyl oxygen

Binding site

121

1

Substrate; via amide nitrogen

Site

122

1

Coordinates a catalytic water molecule

Experimental info

Mutagenesis

121

1

Y → A: 30-fold decrease in catalytic efficiency. Ref.5

Mutagenesis

122

1

E → Q: 700-fold decrease in catalytic efficiency. No ion in metal binding site 1. Ref.5

Mutagenesis

154

1

W → A: Loss of activity. Ref.5

Mutagenesis

154

1

W → F: 340-fold decrease in catalytic efficiency. Ref.5

Mutagenesis

174

1

W → A: Nearly no activity. Ref.5

Mutagenesis

174

1

W → F: 2-fold decrease in catalytic efficiency. Ref.5

Mutagenesis

178

1

H → A: Loss of activity.

Mutagenesis

183

1

E → Q: Loss of activity. Ref.5

Secondary structure

1

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260

Helix Strand Turn

Details...

Helix

7 – 9

3

Helix

12 – 20

9

Beta strand

26 – 30

5

Turn

37 – 39

3

Helix

44 – 60

17

Helix

76 – 79

4

Beta strand

84 – 86

3

Helix

92 – 109

18

Beta strand

113 – 118

6

Helix

121 – 123

3

Helix

124 – 140

17

Beta strand

147 – 152

6

Helix

153 – 156

4

Helix

160 – 166

7

Helix

174 – 176

3

Beta strand

178 – 180

3

Helix

181 – 190

10

Helix

192 – 194

3

Helix

197 – 199

3

Beta strand

210 – 215

6

Helix

218 – 220

3

Helix

235 – 256

22

Sequences

	Sequence		Length	Mass (Da)
	P83772 [UniParc]. Last modified March 15, 2004. Version 1. Checksum: E530A7513F57A762	FASTA	260	28,569
	10 20 30 40 50 60 MSKSVFVGEL TWKEYEARVA AGDCVLMLPV GALEQHGHHM CMNVDVLLPT AVCKRVAERI 70 80 90 100 110 120 GALVMPGLQY GYKSQQKSGG GNHFPGTTSL DGATLTGTVQ DIIRELARHG ARRLVLMNGH 130 140 150 160 170 180 YENSMFIVEG IDLALRELRY AGIQDFKVVV LSYWDFVKDP AVIQQLYPEG FLGWDIEHGG 190 200 210 220 230 240 VFETSLMLAL YPDLVDLDRV VDHPPATFPP YDVFPVDPAR TPAPGTLSSA KTASREKGEL 250 260 ILEVCVQGIA DAIREEFPPT

References

[1]	"Cloning of the creatinine amidohydrolase gene from Pseudomonas sp. PS-7." Yamamoto K., Oka M., Kikuchi T., Emi S. Biosci. Biotechnol. Biochem. 59:1331-1332(1995) [PubMed] [Europe PMC] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA], PROTEIN SEQUENCE OF 2-6. Strain: PS-7.
[2]	"Creatinine amidohydrolase (creatininase) from Pseudomonas putida. Purification and some properties." Rikitake K., Oka I., Ando M., Yoshimoto T., Tsuru D. J. Biochem. 86:1109-1117(1979) [PubMed] [Europe PMC] [Abstract] Cited for: FUNCTION, CATALYTIC ACTIVITY, SUBSTRATE SPECIFICITY, BIOPHYSICOCHEMICAL PROPERTIES, ENZYME REGULATION. Strain: C-83.
[3]	"Crystal structure of creatininase from Pseudomonas putida: a novel fold and a case of convergent evolution." Beuth B., Niefind K., Schomburg D. J. Mol. Biol. 332:287-301(2003) [PubMed] [Europe PMC] [Abstract] Cited for: X-RAY CRYSTALLOGRAPHY (2.10 ANGSTROMS) OF 2-260 IN COMPLEX WITH ZINC, COFACTOR, SUBUNIT.
[4]	"Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism." Yoshimoto T., Tanaka N., Kanada N., Inoue T., Nakajima Y., Haratake M., Nakamura K.T., Xu Y., Ito K. J. Mol. Biol. 337:399-416(2004) [PubMed] [Europe PMC] [Abstract] Cited for: X-RAY CRYSTALLOGRAPHY (1.60 ANGSTROMS) OF APOENZYME AND COMPLEXES WITH CREATINE; ZINC AND MANGANESE, COFACTOR, SUBUNIT, CATALYTIC MECHANISM.
[5]	"Substitution of Glu122 by glutamine revealed the function of the second water molecule as a proton donor in the binuclear metal enzyme creatininase." Yamashita K., Nakajima Y., Matsushita H., Nishiya Y., Yamazawa R., Wu Y.F., Matsubara F., Oyama H., Ito K., Yoshimoto T. J. Mol. Biol. 396:1081-1096(2010) [PubMed] [Europe PMC] [Abstract] Cited for: X-RAY CRYSTALLOGRAPHY (1.78 ANGSTROMS) OF WILD-TYPE IN COMPLEX WITH 1-METHYLGUANIDINE INHIBITOR AND MUTANTS ALA-154; PHE-154; PHE-174 AND GLN-122 IN COMPLEX WITH CREATINE; ZINC AND MANGANESE, CATALYTIC ACTIVITY, KINETIC STUDIES, CATALYTIC MECHANISM, MUTAGENESIS OF TYR-121; GLU-122; TRP-154; TRP-174 AND GLU-183.

Cross-references

Sequence databases

EMBL
GenBank
DDBJ

D45424 Genomic DNA. Translation: BAA08265.1.

PIR

T48846.

3D structure databases

PDBe
RCSB PDB
PDBj

Entry	Method	Resolution (Å)	Chain	Positions	PDBsum
1J2T	X-ray	1.80	A/B/C/D/E/F	1-260	[»]
1J2U	X-ray	1.85	A/B/C/D/E/F	1-260	[»]
1Q3K	X-ray	2.10	A/B/C/D/E/F	2-260	[»]
1V7Z	X-ray	1.60	A/B/C/D/E/F	1-260	[»]
3A6D	X-ray	1.90	A/B/C/D/E/F	1-260	[»]
3A6E	X-ray	2.00	A/B/C/D/E/F	1-260	[»]
3A6F	X-ray	1.78	A/B/C/D/E/F	1-260	[»]
3A6G	X-ray	2.00	A/B/C/D/E/F	1-260	[»]
3A6H	X-ray	2.00	A/B/C/D/E/F	1-260	[»]
3A6J	X-ray	2.00	A/B/C/D/E/F	1-260	[»]
3A6K	X-ray	2.20	A/B/C/D/E/F	1-260	[»]
3A6L	X-ray	2.00	A/B/C/D/E/F	1-260	[»]

ProteinModelPortal

P83772.

SMR

P83772. Positions 3-259.

ModBase

Search...

Protocols and materials databases

StructuralBiologyKnowledgebase

Search...

Enzyme and pathway databases

BioCyc

MetaCyc:MONOMER-10962.

UniPathway

UPA00274.

Family and domain databases

Gene3D

3.40.50.10310. 1 hit.

InterPro

IPR024087. Creatininase-like_dom.
IPR003785. Creatininase/forma_Hydrolase.
[Graphical view]

Pfam

PF02633. Creatininase. 1 hit.
[Graphical view]

SUPFAM

SSF102215. Creatininase. 1 hit.

ProtoNet

Search...

Other

EvolutionaryTrace

P83772.

Entry information

Entry name

CRNA_PSEPU

Accession

Primary (citable) accession number: P83772
Secondary accession number(s): Q52548

Entry history

Integrated into UniProtKB/Swiss-Prot:	April 5, 2011
Last sequence update:	March 15, 2004
Last modified:	April 3, 2013
This is version 36 of the entry and version 1 of the sequence. [Complete history]

Entry status

Reviewed (UniProtKB/Swiss-Prot)

Annotation program

Prokaryotic Protein Annotation Program