The Ntn hydrolases (N-terminal nucleophile) are a diverse superfamily of of enzymes that are activated autocatalytically via an N-terminally lcated nucleophilic amino acid. N-terminal nucleophile (NTN-) hydrolase superfamily, which contains a four-layered alpha, beta, beta, alpha core structure. This family of hydrolases includes penicillin acylase, the 20S proteasome alpha and beta subunits, and glutamate synthase. The mechanism of activation of these proteins is conserved, although they differ in their substrate specificities. All known members catalyze the hydrolysis of amide bonds in either proteins or small molecules, and each one of them is synthesized as a preprotein. For each, an autocatalytic endoproteolytic process generates a new N-terminal residue. This mature N-terminal residue is central to catalysis and acts as both a polarizing base and a nucleophile during the reaction. The N-terminal amino group acts as the proton acceptor and activates either the nucleophilic hydroxyl in a Ser or Thr residue or the nucleophilic thiol in a Cys residue. The position of the N-terminal nucleophile in the active site and the mechanism of catalysis are conserved in this family, despite considerable variation in the protein sequences.

                         10        20        30        40        50        60        70        80
                 ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|
gi 688585456 129 NIFYEVFTVCTSLVAEDNNGNIYHGRNLDFGLFmgwdrqnktwtltEKLKPLVVNINFERKNQTVFKSTSFAGYVGMLTG 208
Cdd:cd01903    1 NIFYEIFTFCTSIVAQDSNGTIYHARNLDFGFF-------------EELSKLTVNVDFQRNGKIVFKGTTFAGYVGLLTG 67

                         90       100
                 ....*....|....*....|.
gi 688585456 209 IRPGELTLTMNERFDFDGGYI 229
Cdd:cd01903   68 QKPGKFSLTINERFSLDGGYN 88

AC_NAAA This conserved domain includes two closely related proteins, acid ceramidase (AC, also known as N-acylsphingosine amidohydrolase), and N-acylethanolamine-hydrolyzing acid amidase (NAAA). AC catalyzes the hydrolysis of ceramide to sphingosine and fatty acid. Ceramide is required for the biosynthesis of most sphingolipids and plays an important role in many signal transduction pathways by inducing apoptosis and/or arresting cell growth. An inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease. AC is considered a "rheostat" important for maintaining the proper intracellular levels of these lipids since hydrolysis of ceramide is the only source of sphingosine in cells. NAAA is a eukaryotic glycoprotein that hydrolyzes bioactive N-acylethanolamines, including anandamide (an endocannabinoid) and N-palmitoylethanolamine (an anti-inflammatory and neuroprotective substance), to fatty acids and ethanolamine at acidic pH. NAAA shows structural and functional similarity to acid ceramidase, but lacks the ceramide-hydrolyzing activity of AC.

                         10        20        30        40        50        60        70        80
                 ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|
gi 688585456 129 NIFYEVFTVCTSLVAEDNNGNIYHGRNLDFGLFmgwdrqnktwtltEKLKPLVVNINFERKNQTVFKSTSFAGYVGMLTG 208
Cdd:cd01903    1 NIFYEIFTFCTSIVAQDSNGTIYHARNLDFGFF-------------EELSKLTVNVDFQRNGKIVFKGTTFAGYVGLLTG 67

                         90       100
                 ....*....|....*....|.
gi 688585456 209 IRPGELTLTMNERFDFDGGYI 229
Cdd:cd01903   68 QKPGKFSLTINERFSLDGGYN 88

C45 family autoproteolytic acyltransferase/hydolase; Members of this family include hydrolases and N-acyltransferases, and belong to the Ntn (N-terminal nucleophile) hydrolase family. Members have an invariant Cys residue (Cys-103 in XP_002569112.1) required both for autoproteolytic processing into alpha and beta chains and for activity. The family is described by MEROPs as a cysteine protease, family C45, because of its autoproteolytic activity. Characterized members include TAN from Drosophila, which removes beta-alanine from both carcinine and N-beta-alanyl dopamine, and isopenicillin-N N-acyltransferase from various fungi. The latter has been heavily studied because of its role in penicillin biosynthesis.

                         10        20        30        40        50        60        70        80
                 ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|
gi 688585456 107 FNEEIKGIAAVSGIPLGEIALFNIFYEVFTV---CTSLVAEDNNGNIYHGRNLDFGlfmgwdrqnktwtltEKLKPLVVN 183
Cdd:NF040521  56 LWEELEGIADGLGLPFEDVLALNARTEILAApdgCSTFAVLGEDGEPILARNYDWH---------------PELYDGCLL 120

                         90       100       110
                 ....*....|....*....|....*....|....*...
gi 688585456 184 INFERKNQTVFKSTSFAGYV-GMLTGI-RPGeLTLTMN 219
Cdd:NF040521 121 LTIRPDGGPRYASIGYAGLLpGRTDGMnEAG-LAVTLN 157

Predicted choloylglycine hydrolase [General function prediction only];

                         10        20        30        40        50
                 ....*....|....*....|....*....|....*....|....*....|.
gi 688585456 109 EEIKGIAAVSGIPLGEIALFNIFYEVFTV-CTSLVAEdNNGNIYHGRNLDF 158
Cdd:COG4927   58 EELEGLADGLDVPLEELLLLNGGYYLPLSgCSQFAVA-PEGEPLLARNYDF 107

AC_NAAA This conserved domain includes two closely related proteins, acid ceramidase (AC, also known as N-acylsphingosine amidohydrolase), and N-acylethanolamine-hydrolyzing acid amidase (NAAA). AC catalyzes the hydrolysis of ceramide to sphingosine and fatty acid. Ceramide is required for the biosynthesis of most sphingolipids and plays an important role in many signal transduction pathways by inducing apoptosis and/or arresting cell growth. An inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease. AC is considered a "rheostat" important for maintaining the proper intracellular levels of these lipids since hydrolysis of ceramide is the only source of sphingosine in cells. NAAA is a eukaryotic glycoprotein that hydrolyzes bioactive N-acylethanolamines, including anandamide (an endocannabinoid) and N-palmitoylethanolamine (an anti-inflammatory and neuroprotective substance), to fatty acids and ethanolamine at acidic pH. NAAA shows structural and functional similarity to acid ceramidase, but lacks the ceramide-hydrolyzing activity of AC.

                         10        20        30        40        50        60        70        80
                 ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|
gi 688585456 129 NIFYEVFTVCTSLVAEDNNGNIYHGRNLDFGLFmgwdrqnktwtltEKLKPLVVNINFERKNQTVFKSTSFAGYVGMLTG 208
Cdd:cd01903    1 NIFYEIFTFCTSIVAQDSNGTIYHARNLDFGFF-------------EELSKLTVNVDFQRNGKIVFKGTTFAGYVGLLTG 67

                         90       100
                 ....*....|....*....|.
gi 688585456 209 IRPGELTLTMNERFDFDGGYI 229
Cdd:cd01903   68 QKPGKFSLTINERFSLDGGYN 88

Choloylglycine hydrolase (CGH)_like. This family of choloylglycine hydrolase-like proteins includes conjugated bile acid hydrolase (CBAH), penicillin V acylase (PVA), acid ceramidase (AC), and N-acylethanolamine-hydrolyzing acid amidase (NAAA) which cleave non-peptide carbon-nitrogen bonds in bile salt constituents. These enzymes have an N-terminal nucleophilic cysteine, as do other members of the Ntn hydrolase family to which they belong. This nucleophilic cysteine is exposed by post-translational prossessing of the precursor protein.

                         10        20        30        40        50        60        70        80
                 ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|
gi 688585456 138 CTSLVAEDNNGNIYHGRNLDFGLFMGwdrqnktwtltekLKPLVVNINFERKNQT---------VFKSTSFAGYVGMLTG 208
Cdd:cd01935    1 CTSIVAQTKDGGVYLGRNMDFSFDYE-------------LRLLVFPRGYQRNGQTgdkskwyakYGSGGTSAGYIGLVDG 67

                         90       100
                 ....*....|....*....|....*.
gi 688585456 209 IRPGELTLTMNERFDFDGGYIDVLYG 234
Cdd:cd01935   68 MNEKGLSVSLLYFPGYAYYPAGIKEG 93

Linear amide C-N hydrolases, choloylglycine hydrolase family; This family includes several hydrolases which cleave carbon-nitrogen bonds, other than peptide bonds, in linear amides. These include choloylglycine hydrolase (conjugated bile acid hydrolase, CBAH) EC:3.5.1.24, penicillin acylase EC:3.5.1.11 and acid ceramidase EC:3.5.1.23. This domain forms the alpha-subunit for members from vertebral species, see family NAAA-beta, pfam15508.

                          10        20        30        40        50        60        70        80
                  ....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|
gi 688585456  138 CTSLVAEDNNGNIYHGRNLDFGLFMGWdrqnktWTLtekLKPLVVNINFER-KNQTVFKstsfAGYVGMLTGIRPGEL-T 215
Cdd:pfam02275   1 CTSITLETKKGNLLFGRNMDFGISYGE------EVI---ITPRNYKLVFEKlGNMLVTK----YAVIGMGTDVGSYPLfY 67

                          90
                  ....*....|....
gi 688585456  216 LTMNER-FDFDGGY 228
Cdd:pfam02275  68 DGLNEKgLGIAGLY 81

Penicillin V acylase (PVA), also known as conjugated bile salt acid hydrolase (CBAH), catalyzes the hydrolysis of penicillin V to yield 6-amino penicillanic acid (6-APA), an important key intermediate of semisynthetic penicillins. PVA has an N-terminal nucleophilic cysteine, as do other members of the Ntn hydrolase family to which PVA belongs. This nucleophilic cysteine is exposed by post-translational prossessing of the PVA precursor. PVA forms a homotetramer.

                         10        20
                 ....*....|....*....|....*..
gi 688585456 138 CTSLVAEDNNGNIYHGRNLDFGLFMGW 164
Cdd:cd00542    1 CTSLTLSTKDGDHVFGRTMDFAFDLGS 27