Substrate specificity of amine oxidase

The tyramine oxidase activity of liver extracts found by Hare (1), the aliphatic amine oxidase activity of brain, kidney, and liver extracts observed by Pugh and Quastel (2), and the adrenalin oxidase activity of similar extracts noted by Blaschko, Richter, and Schlossman (3) were brought under a common enzyme view-point by the latter authors. They were able to show (4) that extracts of brain, instestine, kindey, and liver from a number of mammals or representatives of the birds, reptiles, amphibians, and fishes all acted to absorb oxygen in the presence of several amine substrates. Hare (1) had shown that tyramine and phenethylamine form ammonia in the course of such oxidations, and Richter (5) showed that an ethylamino and a dimethylamino compound, as well as a number of methylamino and amino compounds, all yield the corresponding alkyl-amines or ammonia in the enzymic oxidation. The conslusion that the demonstrated variey of such enzymic activity can be acribed to the presence of a single type pf amine oxidase was dependent in large part on observations that the relative activities of a preparation from one source on a series of substrates bear some relation to the relative activities exhibited by a preparation from another source. Further evidence depended on the action of certain amines as inihibitors and apparent competition between substrates when two oxidizable substrates are present in the system. The degree to which relative activities of different enzyme preparations were constant in a series of substrates was not good in the data reported, and the fact that Hare (1) had not been able to note activity of the liver preparations she used upon adrenalin as the substrate appeared to require special explanations

Substrate Specificity of Peptide Adsorption: A Model Study

Applying the contact density chain-growth algorithm to lattice heteropolymers, we identify the conformational transitions of a nongrafted hydrophobic-polar heteropolymer with 103 residues in the vicinity of a polar, a hydrophobic, and a uniformly attractive substrate. Introducing only two system parameters, the numbers of surface contacts and intrinsic hydrophobic contacts, respectively, we obtain surprisingly complex temperature and solvent dependent, substrate-specific pseudo-phase diagrams.Comment: 5 pages, 2 figure

Structural basis for substrate specificity and regulation of nucleotide sugar transporters in the lipid bilayer

Nucleotide sugars are the activated form of monosaccharides used by glycosyltransferases during glycosylation. In eukaryotes the SLC35 family of solute carriers are responsible for their selective uptake into the Endoplasmic Reticulum or Golgi apparatus. The structure of the yeast GDP-mannose transporter, Vrg4, revealed a requirement for short chain lipids and a marked difference in transport rate between the nucleotide sugar and nucleoside monophosphate, suggesting a complex network of regulatory elements control transport into these organelles. Here we report the crystal structure of the GMP bound complex of Vrg4, revealing the molecular basis for GMP recognition and transport. Molecular dynamics, combined with biochemical analysis, reveal a lipid mediated dimer interface and mechanism for coordinating structural rearrangements during transport. Together these results provide further insight into how SLC35 family transporters function within the secretory pathway and sheds light onto the role that membrane lipids play in regulating transport across the membrane

Evolution of substrate specificity in a recipient's enzyme following horizontal gene transfer

Despite the prominent role of horizontal gene transfer (HGT) in shaping bacterial metabolism, little is known about the impact of HGT on the evolution of enzyme function. Specifically, what is the influence of a recently acquired gene on the function of an existing gene? For example, certain members of the genus Corynebacterium have horizontally acquired a whole L-tryptophan biosynthetic operon, whereas in certain closely related actinobacteria, for example, Mycobacterium, the trpF gene is missing. In Mycobacterium, the function of the trpF gene is performed by a dual-substrate (βα)8 phosphoribosyl isomerase (priA gene) also involved in L-histidine (hisA gene) biosynthesis. We investigated the effect of a HGT-acquired TrpF enzyme upon PriA’s substrate specificity in Corynebacterium through comparative genomics and phylogenetic reconstructions. After comprehensive in vivo and enzyme kinetic analyses of selected PriA homologs, a novel (βα)8 isomerase subfamily with a specialized function in L-histidine biosynthesis, termed subHisA, was confirmed. X-ray crystallography was used to reveal active-site mutations in subHisA important for narrowing of substrate specificity, which when mutated to the naturally occurring amino acid in PriA led to gain of function. Moreover, in silico molecular dynamic analyses demonstrated that the narrowing of substrate specificity of subHisA is concomitant with loss of ancestral protein conformational states. Our results show the importance of HGT in shaping enzyme evolution and metabolism

The Unusual Substrate Specificity of a Virulence Associated Serine Hydrolase from the Highly Toxic Bacterium, \u3cem\u3eFrancisella tularensis\u3c/em\u3e

Francisella tularensis is the causative agent of the highly, infectious disease, tularemia. Amongst the genes identified as essential to the virulence of F. tularensis was the proposed serine hydrolase FTT0941c. Herein, we purified FTT0941c to homogeneity and then characterized the folded stability, enzymatic activity, and substrate specificity of FTT0941c. Based on phylogenetic analysis, FTT0941c was classified within a divergent Francisella subbranch of the bacterial hormone sensitive lipase (HSL) superfamily, but with the conserved sequence motifs of a bacterial serine hydrolase. FTT0941c showed broad hydrolase activity against diverse libraries of ester substrates, including significant hydrolytic activity across alkyl ester substrates from 2 to 8 carbons in length. Among a diverse library of fluorogenic substrates, FTT0941c preferred α-cyclohexyl ester substrates, matching with the substrate specificity of structural homologues and the broad open architecture of its modeled binding pocket. By substitutional analysis, FTT0941c was confirmed to have a classic catalytic triad of Ser115, His278, and Asp248 and to remain thermally stable even after substitution. Its overall substrate specificity profile, divergent phylogenetic homology, and preliminary pathway analysis suggested potential biological functions for FTT0941c in diverse metabolic degradation pathways in F. tularensis

Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding

Two mutant forms of penicillin acylase from Escherichia coli strains, selected using directed evolution for the ability to use glutaryl-L-leucine for growth [Forney, Wong and Ferber (1989) Appl. Environ. Microbiol. 55, 2550-2555], are changed within one codon, replacing the B-chain residue Phe(B71) with either Cys or Leu. Increases of up to a factor of ten in k(cat)/K-m values for substrates possessing a phenylacetyl leaving group are consistent with a decrease in K-s. Values of k(cat/)K(m) for glutaryl-L-leucine are increased at least 100-fold. A decrease in k(cat)/K-m for the CySB71 mutant with increased pH is consistent with binding of the uncharged glutaryl group. The mutant proteins are more resistant to urea denaturation monitored by protein fluorescence, to inactivation in the presence of substrate either in the presence of urea or at high pH, and to heat inactivation. The crystal structure of the Leu(B71) mutant protein, solved to 2 X resolution, shows a flip of the side chain of Phe(B256) into the periphery of the catalytic centre, associated with loss of the pi-stacking interactions between Phe(B256) and Phe(B71). Molecular modelling demonstrates that glutaryl-L-leucine may bind with the uncharged glutaryl group in the S-1 subsite of either the wild-type or the Leu(B71) mutant but with greater potential freedom of rotation of the substrate leucine moiety in the complex with the mutant protein. This implies a smaller decrease in the conformational entropy of the substrate on binding to the mutant proteins and consequently greater catalytic activity

Substrate specificity and the effect of calcium on Trypanosomabrucei metacaspase 2

Metacaspases are cysteine peptidases found only in yeast, plants and lower eukaryotes, including the protozoa. To investigate the extended substrate specificity and effects of Ca<sup>2+</sup> on the activation of these enzymes, detailed kinetic, biochemical and structural analyses were carried out on metacaspase 2 from Trypanosoma brucei (TbMCA2). These results reveal that TbMCA2 has an unambiguous preference for basic amino acids at the P<sub>1</sub> position of peptide substrates and that this is most probably a result of hydrogen bonding from the P<sub>1</sub> residue to Asp95 and Asp211 in TbMCA2. In addition, TbMCA2 also has a preference for charged residues at the P<sub>2</sub> and P<sub>3</sub>positions and for small residues at the prime side of a peptide substrate. Studies into the effects of Ca<sup>2+</sup> on the enzyme revealed the presence of two Ca<sup>2+</sup> binding sites and a reversible structural modification of the enzyme upon Ca<sup>2+</sup> binding. In addition, the concentration of Ca<sup>2+</sup> used for activation of TbMCA2 was found to produce a differential effect on the activity of TbMCA2, but only when a series of peptides that differed in P<sub>2</sub> were examined, suggesting that Ca<sup>2+</sup>activation of TbMCA2 has a structural effect on the enzyme in the vicinity of the S2 binding pocket. Collectively, these data give new insights into the substrate specificity and Ca<sup>2+</sup> activation of TbMCA2. This provides important functional details and leads to a better understanding of metacaspases, which are known to play an important role in trypanosomes and make attractive drug targets due to their absence in humans

Structural basis for substrate specificity of heteromeric transporters of neutral amino acids

Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo-electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies