Structural insights into the catalysis and substrate specificity of cyanobacterial aspartate racemase McyF

Through the Looking Glass: Chiral Recognition of Substrates and Products at the Active Sites of Racemases and Epimerases

“…[261,262] That these enzymes already possess the ability to bind enantiomersw ith the same orientations as the substrates of racemases and epimerases is intriguing since it suggests that these enzymes might be amenable to engineering of racemase or epimerase activity by rational designa nd/ord irected evolution with, of course, concomitant reduction or obviation of their native activity. [a] alaniner acemase (AlaR) 1L6F (PLP-l-Ala, 2.0 ) [69] 1L6G (PLP-d-Ala, 2.0 ) [69] 2VD9 (PLP-l-AlaPand PLP-d-Ala, 2.1 ) [71] 1.02 1.33 mirror isoleucine 2-epimerase (IleE)5WYA(PLP-l-isoleucine,2 .65 ) [74] 5WYF (PLP-d-allo-isoleucine, 2.12 ) [74] 0.75 mirror serine racemase (SerR) 2ZR8 (serine, 2.2 )model [81] 0.48 super/NS a-amino-e-caprolactam racemase (ACLR) PDB 5M49(l-a nd d-a-amino-e-caprolactam, 1.51 ) [83] 1.43 mirror/NS glutamate racemase( GluR) 2JFO (l-glutamate and d-glutamate, 2.5 ) [84] - [b] possibly super aspartater acemase (AspR) 5WXY (l-aspartate, 2.63 ) [85] 5WXZ (d-aspartate, 2.80 ) [85] 1.00 mirror Asp/Glu racemase 5HRC (l-aspartate, 1.76 ) [86] 5HRA (d-aspartate, 1.60 ) [86] 1.16 mirror diaminopimelate epimerase (DAPE) 2GKJ (d,l-aziridino-DAP, 1.55 ) [87] 2GKE (l,l-aziridino-DAP,1.70 ) [87] 3EKM (d,l-aziridino-DAP,1.95 ) [88] 3EJX (l,l-aziridino-DAP,2 .30 ) [88] 0.87 0.54 mirror [a] proliner acemase(ProR) 1W61 (pyrrole-2-carboxylate, 2.10 ) [136] -NS l-Ala-d/l-Glud ipeptide epimerase (AEE) 3R1Z (l-Ala-l-Glua nd l-Ala-d-Glu, 2.9 ) [89] 1.12 [b] mirror histidine racemase (HisR) model based on 5ZL6 (PLP,2 .10 ) [142] model based on 6JIS (apo, 1.82 ) [90] 1.67 [b] mirror a-methylacyl-coenzyme Aracemase (epimerase) (AMACR) 2GCE ((2S)-ibuprofenoyl-CoA and (2R)-ibuprofenoyl-CoA, 1.85 ) [91] 2GD0((2S)-methylmyristoyl-CoA, 1.70 ) [91] 2GCI ((2R)-methylmyristoyl-CoA, 1.60 ) [91] 0.98 0.81 mirror mandelate racemase (MR)3UXK (benzohydroxamate, 2.20 ) [92] 3UXL (Cupferro...…”

“…[84] (G) l-Asp (blue) and d-Asp( green) as bound to AspRf rom Microcystis aeruginosa PCC 7806 (PDB 5WXYand 5WXZ, respectively) are shown. [85] (H) l-Asp (blue) and d-Asp (green)a sb ound to Asp/Glu racemase from Escherichia coli O157( PDB 5HRC and 5HRA, respectively) are shown. [86] (I) The l,l-epimer (blue) and d,l-epimer (green)ofa ziridino-DAP( i.e.,azi-DAP,a ni rreversiblei nhibitor and substrate analogue) are showncovalently linked to sulfur atomso f the d,l-and l,l-specific Brønsted basec atalysts Cys217a nd Cys73ofD APE from Haemophilus influenzae,respectively (PDB 2GKJ and 2GKE, respectively).…”

“…[48] This d-amino acid is also found in the peptidoglycan cross-bridges, [123,124] as well as in several natural antibiotics (e.g.,b acitracin), antifun-gals (e.g.,m ycobacillin), and toxins (e.g.,m icrocystin). [48,85] Recently,k inetic and structurals tudies were reported for the AspR produced by the cyanobacterium Microcystis aeruginosa PCC7806,w hich furnishes the d-Asp required for the biosynthesis of microcystin. [85] AspR (EC 5.1.1.13) is aPLP-independent racemase that utilizes at wo-base mechanism with Cys195 and Cys87 acting as the enantiospecific Brønsted bases to abstract the a-proton from l-and d-Asp, respectively (Scheme5).…”

“…[48,85] Recently,k inetic and structurals tudies were reported for the AspR produced by the cyanobacterium Microcystis aeruginosa PCC7806,w hich furnishes the d-Asp required for the biosynthesis of microcystin. [85] AspR (EC 5.1.1.13) is aPLP-independent racemase that utilizes at wo-base mechanism with Cys195 and Cys87 acting as the enantiospecific Brønsted bases to abstract the a-proton from l-and d-Asp, respectively (Scheme5). In addition, the enzymea lso appearst oc atalyze the slow interconversion of l-a nd d-b-methylaspartate.…”

“…In addition, the enzymea lso appearst oc atalyze the slow interconversion of l-a nd d-b-methylaspartate. [85,125,126] The enzyme is a homodimer in solution and each subunit is comprised of two domainsw ith the active site located in ac left between the domains.C omparison of the superposed structures of the bound substrates ( Figure 2G)o ft he C87S/C195S variant complexed with l-Asp (PDB 5WXY) and d-Asp (PDB 5WXZ) reveals that each pair of the carboxylate groups and the amino groups of the enantiomers interact with the same binding determinants at the active site. [85] The a-COO À is held in place by an H-bond with the side chain of Thr89;t he b-COO À is held in place by Hbonds with the side chains of Arg53, Ser87,a nd Lys167;a nd the a-NH 3…”