title: Structural insights into the γ-lactamase activity and substrate enantioselectivity of an isochorismatase-like hydrolase from Microbacterium hydrocarbonoxydans.
abstract: (+)-γ-lactamase catalyzes the specific hydrolysis of (+)-γ-lactam out of the racemic γ-lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one) to leave optically pure (-)-γ-lactam, which is the key building block of antiviral drugs such as carbovir and abacavir. However, no structural data has been reported on how the enzymes bind the γ-lactams and achieve their enantioselectivities. We previously identified an isochorismatase-like hydrolase (IHL, Mh33H4-5540) with (+)-γ-lactamase activity, which constitutes a novel family of γ-lactamase. Here, we first discovered that this enzyme actually hydrolyzed both (+)- and (-)-γ-lactam, but with apparently different specificities. We determined the crystal structures of the apo-form, (+)-γ-lactam bound, and (-)-γ-lactam bound forms of the enzyme. The structures showed that the binding sites of both (+) and (-)-γ-lactam resemble those of IHLs, but the "cover" loop conserved in IHLs is lacking in the enzyme, probably resulting in its incomplete enantioselectivity. Structural, biochemical, and molecular dynamics simulation studies demonstrated that the steric clash caused by the binding-site residues, especially the side-chain of Cys111 would reduce the binding affinity of (-)-γ-lactam and possibly the catalytic efficiency, which might explain the different catalytic specificities of the enantiomers of γ-lactam. Our results would facilitate the directed evolution and application of Mh33H4-5540 in antiviral drug synthesis.
authors.affiliation: Beijing Key Lab of Bioprocess, the State Key Laboratory of Chemical Resources Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China.
authors.forename: Shuaihua
authors.initials: S
authors.lastname: Gao
authors.affiliation: National Institute of Biological Sciences, Beijing, No. 7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, PR China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Beijing Key Lab of Bioprocess, the State Key Laboratory of Chemical Resources Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Beijing Key Lab of Bioprocess, the State Key Laboratory of Chemical Resources Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China.
authors.forename: Wenhe
authors.initials: W
authors.lastname: Wang
authors.affiliation: Key Laboratory for Protein Sciences of Ministry of Education, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, 100084, Beijing, PR China.
authors.forename: You
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Beijing Key Lab of Bioprocess, the State Key Laboratory of Chemical Resources Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China.
authors.forename: Yajuan
authors.initials: Y
authors.lastname: Mu
authors.affiliation: Beijing Key Lab of Bioprocess, the State Key Laboratory of Chemical Resources Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China.
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.affiliation: Institute for Mathematical Sciences, Renmin University of China, Beijing 100872, PR China.
authors.forename: Xinqi
authors.initials: X
authors.lastname: Gong
authors.affiliation: Beijing Key Lab of Bioprocess, the State Key Laboratory of Chemical Resources Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China.
authors.forename: Guojun
authors.initials: G
authors.lastname: Zheng
authors.affiliation: Beijing Key Lab of Bioprocess, the State Key Laboratory of Chemical Resources Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Feng
mesh_terms: D000192:Actinomycetales / Q000201:enzymology; D000581:Amidohydrolases / Q000737:chemistry* / Q000648:ultrastructure; D001665:Binding Sites; D006867:Hydrolases / Q000737:chemistry* / Q000648:ultrastructure; D006868:Hydrolysis; D007769:Lactams / Q000737:chemistry*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D007769:Lactams; D006867:Hydrolases; C064012:isochorismatase; D000581:Amidohydrolases; C401943:gamma-lactamase