Structural and Functional Characterization of Enzymes in COG3964 of the Amidohydrolase Superfamily: From Sequence to Structure to Function

• Main Author: Ornelas, Argentina 1982-
• Other Authors: Raushel, Frank M
• Format: Others
• Published: 2013
• Subjects: protein clusters; COG3964; functional misannotations; enzyme superfamilies; amidohydrolase
• Online Access: http://hdl.handle.net/1969.1/148173

The Amidohydrolase Superfamily (AHS) of enzymes is one of the most structurally and functionally studied groups of biological catalysts, exquisitely designed to carry out an extensive number of reactions defined by a similar reaction mechanism. There are approximately 11,000 genes coding for AHS proteins from about 2,100 sequenced organisms. Sequence information for these genes has been catalogued in databases, the most instrumental being the National Center for Biotechnology Information (NCBI). Despite the accessible information organized in genomic databases, there is still an extensive problem of reliability in the functional annotation of gene products assigned to the AHS. Proteins in COG3964 of the AHS have been functionally identified as dihydroorotases and adenine deaminases. Eight proteins within three group families of COG3964 have been purified and fail to demonstrate the functionally annotated activity. A library of compounds developed by functional-group modifications was compiled and tested with these enzymes. A group of enzymes within COG3964 demonstrates the ability to hydrolyze stereospecific acetylated alpha-hydroxyl carboxylates. Substrate profiles were constructed for enzymes belonging to group 6 of COG3964. Atu3266, Oant2987 and RHE_PE00295 hydrolyze the R-isomers of a library of alpha-acetyl carboxylates of which acetyl-R-mandelate is the best substrate with catalytic efficiencies of 10^5 M^-1s^-1. This compound was identified after a series of modifications from a low-activity compound (V/K = 4 M^-1s^-1). Methylphosphonate analogs of acetyl-R-mandelate and N-acetyl-D-phenyl glycine are inhibitors of enzymes in group 6. The structure of Atu3266 was used in docking experiments to assess the selectivity of R- enantiomers over their S- counterparts. An additional group of orthologues share less than 40% sequence similarity to enzymes from group 6. EF0837, STM4445 and BCE_5003 from group 2 show significantly lower rates for the hydrolysis of alpha-acetyl carboxylates, including acetyl-R-mandelate, hydrolyzed at values of kcat/Km = 10^3 M^-1s^-1. This is also the only active compound for EF0837. Xaut_0650 and blr3349 from group 7 of COG3964 demonstrate less than 30% identity to enzymes in groups 2 and 6. These enzymes fail to hydrolyze any compound from an extended library of compounds. An annotated selenocysteine synthase gene (SelA) from COG1921 has been identified as a gene neighbor to almost every amidohydrolase from COG3964. Atu3263, Oant2990 and EF0838 are pyridoxal-5’-phosphate dependent enzymes that were purified and assayed with D- and L- amino acids. Initial thermal-shift fluorescence assays determined that in the presence of D-cysteine, the proteins were denatured at lower temperatures.