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Literature summary for 3.5.2.B2 extracted from

  • Gao, S.; Zhou, Y.; Zhang, W.; Wang, W.; Yu, Y.; Mu, Y.; Wang, H.; Gong, X.; Zheng, G.; Feng, Y.
    Structural insights into the ?-lactamase activity and substrate enantioselectivity of an isochorismatase-like hydrolase from Microbacterium hydrocarbonoxydans (2017), Sci. Rep., 7, 44542 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.5.2.B2

Application

Application Comment Organism
drug development application of the enzyme in antiviral drug synthesis. The enzyme catalyzes the specific hydrolysis of (+)-gamma-lactam out of the racemic gamma-lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) to leave optically pure (-)-gamma-lactam, which is the key building block of antiviral drugs such as carbovir and abacavir Microbacterium hydrocarbonoxydans
synthesis (+)-gamma-lactamase catalyzes the specific hydrolysis of (+)-gamma-lactam out of the racemic gamma-lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) to leave optically pure (-)-gamma-lactam, which is the key building block of antiviral drugs such as carbovir and abacavir Microbacterium hydrocarbonoxydans

Cloned(Commentary)

Cloned (Comment) Organism
DNA and amino acid sequence determination and analysis, sequence comparisons Microbacterium hydrocarbonoxydans
expression in Escherichia coli Rosetta (DE3) Microbacterium hydrocarbonoxydans

Crystallization (Commentary)

Crystallization (Comment) Organism
purified apo-form, (+)-gamma-lactam bound, and (-)-gamma-lactam bound forms of the enzyme, X-ray diffraction structure determination and analysis at 1.79-2.05 A resolution Microbacterium hydrocarbonoxydans
sitting drop vapor diffusion method at 20°C, crystal structures of the apo-form, (+)-gamma-lactam bound, and (-)-gamma-lactam bound forms of the enzyme Microbacterium hydrocarbonoxydans

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
12.9
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one pH 7.5, 25°C Microbacterium hydrocarbonoxydans
34.2
-
 (-)-2-azabicyclo[2.2.1]hept-5-en-3-one pH 7.5, 25°C Microbacterium hydrocarbonoxydans

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
(1S,4R)-2-azabicyclo[2.2.1]hept-5-en-3-one + H2O Microbacterium hydrocarbonoxydans
-
 (1R,4S)-4-aminocyclopent-2-ene-1-carbaldehyde
-
 ?

Organism

Organism UniProt Comment Textmining
Microbacterium hydrocarbonoxydans A0A0K0XHU0
-
-
Microbacterium hydrocarbonoxydans E3SVR9
-
-

Purification (Commentary)

Purification (Comment) Organism
-
 Microbacterium hydrocarbonoxydans

Reaction

Reaction Comment Organism Reaction ID
a (+)-gamma-lactam + H2O = a substituted gamma-amino acid reaction mechanism, overview Microbacterium hydrocarbonoxydans
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(1S,4R)-2-azabicyclo[2.2.1]hept-5-en-3-one + H2O
-
 Microbacterium hydrocarbonoxydans (1R,4S)-4-aminocyclopent-2-ene-1-carbaldehyde
-
 ?
2-azabicyclo[2.2.1]hept-5-en-3-one + H2O low activity with (-)-2-azabicyclo[2.2.1]hept-5-en-3-one, high activity with (+)-2-azabicyclo[2.2.1]hept-5-en-3-one Microbacterium hydrocarbonoxydans (-)-2-azabicyclo[2.2.1]hept-5-en-3-one + (+)-4-amino-cyclopent-2-enecarboxylic acid
-
 ?
2-azabicyclo[2.2.1]hept-5-en-3-one + H2O low activity with (-)-2-azabicyclo[2.2.1]hept-5-en-3-one, high activity with (+)-2-azabicyclo[2.2.1]hept-5-en-3-one Microbacterium hydrocarbonoxydans (+)-2-azabicyclo[2.2.1]hept-5-en-3-one + (-)-4-amino-cyclopent-2-enecarboxylic acid
-
 ?
additional information the enzyme hydrolyzes both (+)- and (-)-gamma-lactam, but with apparently different specificities. Enantioselectivity of the reaction, overview. Enzyme MhIHL preferentially hydrolyzes (+)-gamma-lactam versus (-)-gamma-lactam. The active site of MhIHL is located at the C-termini of the six-stranded beta-sheet, similar to other known structures with substrates. A clear electron density for one ligand is observed in the active site of each protomer in both (+)- and (-)-gamma-lactam-bound MhIHL structures. (+)-gamma-Lactam and (-)-gamma-lactam bind to nearly the same position in MhIHL Microbacterium hydrocarbonoxydans ?
-
 ?

Synonyms

Synonyms Comment Organism
(+)-gamma-lactamase
-
 Microbacterium hydrocarbonoxydans
IHL
-
 Microbacterium hydrocarbonoxydans
isochorismatase-like hydrolase
-
 Microbacterium hydrocarbonoxydans
Mh33H4-5540
-
 Microbacterium hydrocarbonoxydans
MhIHL
-
 Microbacterium hydrocarbonoxydans

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Microbacterium hydrocarbonoxydans

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
19.8
-
 (-)-2-azabicyclo[2.2.1]hept-5-en-3-one pH 7.5, 25°C Microbacterium hydrocarbonoxydans
203.4
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one pH 7.5, 25°C Microbacterium hydrocarbonoxydans

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Microbacterium hydrocarbonoxydans

General Information

General Information Comment Organism
evolution the isochorismatase-like hydrolase (IHL, Mh33H4-5540, EC 3.3.2.1) with (+)-gamma-lactamase activity constitutes a distinct family of gamma-lactamase Microbacterium hydrocarbonoxydans
additional information the enzyme crystal structures show that the binding sites of both (+) and (-)-gamma-lactam resemble those of isochorismatase-like hydrolases, but the cover loop conserved in isochorismatase-like hydrolases is lacking in the enzyme, probably resulting in its incomplete enantioselectivity. Structural, biochemical, and molecular dynamics simulation studies demonstrate that the steric clash caused by the binding-site residues, especially the side-chain of Cys111 reduces the binding affinity of (-)-gamma-lactam and possibly the catalytic efficiency, which might explain the different catalytic specificities of the enantiomers of gamma-lactam Microbacterium hydrocarbonoxydans
physiological function (+)-gamma-lactamase catalyzes the specific hydrolysis of (+)-gamma-lactam out of the racemic gamma-lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) to leave optically pure (-)-gamma-lactam, which is the key building block of antiviral drugs such as carbovir and abacavir Microbacterium hydrocarbonoxydans

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.58
-
 (-)-2-azabicyclo[2.2.1]hept-5-en-3-one pH 7.5, 25°C Microbacterium hydrocarbonoxydans
15.77
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one pH 7.5, 25°C Microbacterium hydrocarbonoxydans