Crystal Structure of the Leucine Aminopeptidase from Pseudomonas putida Reveals the Molecular Basis for its Enantioselectivity and Broad Substrate Specificity

Kale, Avinash; Pijning, Tjaard; Sonke, Theo; Dijkstra, Bauke W.; Thunnissen, A.M.W.H.

doi:10.1016/j.jmb.2010.03.042

Cited by 30 publications

(28 citation statements)

References 35 publications

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“…No activity could be detected, which we believed to be a result of the absence of metal ions (as seen in the structure). Typical metals found in active LAPs include Zn 2+ , Mn 2+ , Co 2+ and Mg 2+ ions . The proteolysis reaction was repeated with a truncated version of BotA in the presence of 100 μ m of each metal ion and reaction progress was monitored over time using LC‐MS (Figure A).…”

Section: Resultsmentioning

confidence: 99%

Structure and Substrate Recognition of the Bottromycin Maturation Enzyme BotP

et al. 2016

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Abstract:The bottromycins are a family of highly modified peptide natural products displaying potent antimicrobial activity against Gram-positive bacteria including methicillin-resistant Staphyloccoccus aureus. Bottromycins have recently been shown to be ribosomally synthesized and post-translationally modified peptides (RiPPs). Uniquely amongst RiPPs the precursor peptide BotA contains a C-terminal follower, rather than the canonical Nterminal leader sequence. We report the structural and biochemical characterization of BotP, a leucyl-aminopeptidase like enzyme from the bottromycin pathway. We demonstrate that BotP is responsible for the removal of the N-terminal methionine from the precursor peptide. The crystal structures of apo BotP and of BotP in complex with Mn 2+ allowed us to model a BotP/substrate complex and to rationalize substrate recognition. Our data represent the first step towards targeted compound modification to unlock the full antibiotic potential of bottromycin.

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Section: Resultsmentioning

confidence: 99%

Structure and Substrate Recognition of the Bottromycin Maturation Enzyme BotP

et al. 2016

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“…Aminopeptidases are metalloproteinases that cleave N‐terminal residues from proteins and small oligopeptides. The enzymes are widely distributed in nature and play crucial roles in several important physiological processes, including protein degradation and turnover, the metabolism of biologically active peptides, and generation of metabolic energy . An aminopeptidase P (PepP) liberates N‐terminal amino acids when proline is in the penultimate position and displays a specificity for Xaa‐Pro‐Pro‐(Yaa) n sequences .…”

Section: Discussionmentioning

confidence: 99%

Genes involved in nutrient competition by Pseudomonas putida JBC17 to suppress green mold in postharvest satsuma mandarin

Lee

2015

J. Basic Microbiol.

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Understanding the mechanisms underlying biocontrol activity in biocontrol agents is indispensable to implement biological control. However, the understanding of specific mechanisms for nutrient competition in nutrient limited environments is still limited. This study was performed to control green mold of postharvest satsuma mandarin (mandarin) using Pseudomonas putida JBC17 (JBC17), and identify the genes involved in nutrient competition. Treatment with JBC17 on wounded mandarin fruits at a concentration of 10(6) and 10(7) cfu ml(-1) suppressed the incidence of green mold with efficacy of 74.1 and 91.4%, respectively, compared to the untreated control. In spite of there being no antifungal activity in a dual culture test, JBC17 significantly inhibited the conidial germination of Penicillium digitatum. The results from the nutrient competition assay revealed that the inhibition of conidial germination was exerted by nutrient starvation. From the constructed transposon (Tn) library of JBC17, exopolyphosphatase (ppx) and Xaa-Pro aminopeptidase (pepP) were recognized as potential factors responsible for the inhibition of conidial germination. In conclusion, the understanding of nutrient depletion specific to the inhibition of conidial germination by JBC17 may ultimately lead to a deeper understanding of the bacterial metabolism and conidial metabolism for germination as well as biocontrol activity.

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“…Crystal structures of several LAP enzymes have been determined, including those from Bos taurus (Kraft et al, 2006;Strä ter, Sun et al, 1999), E. coli (Strä ter, Sherratt et al, 1999), Pseudomonas putida (Kale et al, 2010) and Plasmodium falciparum (McGowan et al, 2010), all of which exhibit a hexameric structure with bilobal monomer subunits. From these structures, key residues were identified that facilitate metal-cofactor and substrate binding and catalysis (Gu & Walling, 2002;Kraft et al, 2006;Strä ter, Sherratt et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

“…The oxyanion is further stabilized by an interaction with the nearby Lys residue. The bicarbonate ion is believed to act as a general base, abstracting a proton from the nucleophilic water molecule and transferring it to the amino-terminal group of the P1 0 product after cleavage of the peptide bond (Kale et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Structure of tomato wound-induced leucine aminopeptidase sheds light on substrate specificity

DuPrez

Scranton

Walling

et al. 2014

Acta Cryst D Biol Crystallogr

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The acidic leucine aminopeptidase (LAP-A) from tomato is induced in response to wounding and insect feeding. Although LAP-A shows in vitro peptidase activity towards peptides and peptide analogs, it is not clear what kind of substrates LAP-A hydrolyzes in vivo. In the current study, the crystal structure of LAP-A was determined to 2.20 Å resolution. Like other LAPs in the M17 peptidase family, LAP-A is a dimer of trimers containing six monomers of bilobal structure. Each monomer contains two metal ions bridged by a water or a hydroxyl ion at the active site. Modeling of different peptides or peptide analogs in the active site of LAP-A reveals a spacious substrate-binding channel that can bind peptides of five or fewer residues with few geometric restrictions. The sequence specificity of the bound peptide is likely to be selected by the structural and chemical restrictions on the amino acid at the P1 and P1' positions because these two amino acids have to bind perfectly at the active site for hydrolysis of the first peptide bond to occur. The hexameric assembly results in the merger of the open ends of the six substrate-binding channels from the LAP-A monomers to form a spacious central cavity allowing the hexameric LAP-A enzyme to simultaneously hydrolyze six peptides containing up to six amino acids each. The hexameric LAP-A enzyme may also hydrolyze long peptides or proteins if only one such substrate is bound to the hexamer because the substrate can extend through the central cavity and the two major solvent channels between the two LAP-A trimers.

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Crystal Structure of the Leucine Aminopeptidase from Pseudomonas putida Reveals the Molecular Basis for its Enantioselectivity and Broad Substrate Specificity

Cited by 30 publications

References 35 publications

Structure and Substrate Recognition of the Bottromycin Maturation Enzyme BotP

Structure and Substrate Recognition of the Bottromycin Maturation Enzyme BotP

Genes involved in nutrient competition by Pseudomonas putida JBC17 to suppress green mold in postharvest satsuma mandarin

Structure of tomato wound-induced leucine aminopeptidase sheds light on substrate specificity

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