Aminopeptidase from Sphingomonas capsulata

Abstract

A novel aminopeptidase with unique substrate specificity was purified from a culture broth of Sphingomonas capsulata. This is the first reported aminopeptidase to demonstrate broad substrate specificity and yet release glycine and alanine with the highest efficacy. On a series of pentapeptide amides with different N-terminal amino acids, this enzyme efficiently releases glycine, alanine, leucine, proline, and glutamate with the lowest turnover value of 370 min ؊1 for glutamate. At pH 7.5 (pH optimum) and 25°C, the kinetic parameters for alanine para-nitroanilide were found to be k cat ‫؍‬ 7600 min ؊1and K m ‫؍‬ 14 mM. For alanine ␤-naphthylamide, they were k cat ‫؍‬ 860 min ؊1 and K m ‫؍‬ 6.7 mM. Polymerase chain reaction primers were designed based upon obtained internal sequences of the wild type enzyme. The subsequent product was then used to acquire the fulllength gene from an S. capsulata genomic library. An open reading frame encoding a protein of 670 amino acids was obtained. The translated protein has a putative signal peptide that directs the enzyme into the supernatant. A search of the amino acid sequence revealed no significant homology to any known aminopeptidases in the available data bases.The rising interest, both basic and applied, in aminopeptidases (EC 3.4.11) from different sources has led to the discovery of a number of enzymes that differ from each other in cellular location, catalytic mechanism, and substrate specificity (1-3). The majority of bacterial monoaminopeptidases are intracellular or membrane-bound metalloenzymes (1). Based on substrate specificity, bacterial monoaminopeptidases can be divided into two basic categories, specific aminopeptidases, which release only a limited number of amino acids, and those that are able to liberate a relatively broad spectrum of Nterminal amino acid residues (1).Proline and glycine are among the most difficult residues for aminopeptidases to hydrolyze because of their unique structures. Proline is unusual because of its cyclic structure, and glycine is identified by the lack of a side chain. Nature has developed a family of enzymes that recognize proline exclusively (4). A monoaminopeptidase that preferentially releases glycine with high efficiency has not yet been described and thus would be of high interest.In this report we describe a novel extracellular monoaminopeptidase from Sphingomonas capsulata. This enzyme has a clear preference for N-terminal glycine and alanine. Because of this characteristic, this monoaminopeptidase has the potential to significantly enhance the degree of protein hydrolysis (5) when used as a supplement to endoproteases and other exopeptidases. EXPERIMENTAL PROCEDURES MaterialsChemicals used as buffers and reagents were commercial products of at least reagent grade. para-Nitroanilides of L-amino acids and peptide substrates were from Sigma or Bachem. Pentapeptide amides were synthesized at the Core Laboratories (Louisiana State University). S. capsulata strain IFO 12533 was purchased from the Institute for Fermentatio...

trans-2-Phenylcyclopropylamine Is a Mechanism-Based Inactivator of the Histone Demethylase LSD1

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Characterization of a Glycyl-Specific TET Aminopeptidase Complex from Pyrococcus horikoshii

“…While aminopeptidases from different families are capable of hydrolyzing glycine residues, their activities remain weak in comparison with their preferred substrates. To our knowledge, only three aminopeptidases have been found to exhibit clear preferences for glycine residues; the first is a Zn-dependent metallopeptidase from the M61 family secreted by the Gram-negative bacterium Sphingomonas capsulata (30), the second is a eukaryotic S12-family serine peptidase found in the cytosol of Actinomucor oryzae (20), and the third is the cytosolic GAP of Actinomucor elegans (31), for which the residues implicated in the enzymatic mechanism are still ambiguous. Therefore, PhTET4 is the first GAP enzyme identified in archaea.…”

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“…The most striking difference between the two enzymes was observed with respect to their specificity toward synthetic as well as peptide substrates. ScapAP exhibited a broad specificity for synthetic substrates with a preference for N‐terminal Ala, followed by Leu>Met>Gly>Asp>Glu (Byun et al , 2001; Byun & Blinkovsky, 2004), while BcepAP preferentially hydrolyzed Asp residues and demonstrated insignificant activity (≤3%) toward N‐terminal Ala, Gly or Ser residues. However, when peptide substrates were used, ScapAP showed a preference for Gly, followed by Ala>Leu>Glu>Pro, while BcepAP exhibited a preference either for the Asp or for the Glu residue.…”

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