<i>Streptomyces wadayamensis</i> MppP Is a Pyridoxal 5′-Phosphate-Dependent l‑Arginine α‑Deaminase, γ‑Hydroxylase in the Enduracididine Biosynthetic Pathway

l-Enduracididine (l-End) is a nonproteinogenic amino acid found in a number of bioactive peptides, including the antibiotics teixobactin, enduracidin, and mannopeptimycin. The potent activity of these compounds against antibiotic-resistant pathogens like MRSA and their novel mode of action have garnered considerable interest for the development of these peptides into clinically relevant antibiotics. This goal has been hampered, at least in part, by the fact that l-End is difficult to synthesize and not currently commercially available. We have begun to elucidate the biosynthetic pathway of this unusual building block. In mannopeptimycin-producing strains, like <i>Streptomyces wadayamensis</i>, l-End is produced from l-Arg by the action of three enzymes: MppP, MppQ, and MppR. Herein, we report the structural and functional characterization of MppP. This pyridoxal 5′-phosphate (PLP)-dependent enzyme was predicted to be a fold type I aminotransferase on the basis of sequence analysis. We show that MppP is actually the first example of a PLP-dependent hydroxylase that catalyzes a reaction of l-Arg with dioxygen to yield a mixture of 2-oxo-4-hydroxy-5-guanidinovaleric acid and 2-oxo-5-guanidinovaleric acid in a 1.7:1 ratio. The structure of MppP with PLP bound to the catalytic lysine residue (Lys221) shows that, while the tertiary structure is very similar to those of the well-studied aminotransferases, there are differences in the arrangement of active site residues around the cofactor that likely account for the unusual activity of this enzyme. The structure of MppP with the substrate analogue d-Arg bound shows how the enzyme binds its substrate and indicates why d-Arg is not a substrate. On the basis of this work and previous work with MppR, we propose a plausible biosynthetic scheme for l-End

<i>Streptomyces wadayamensis</i> MppP is a PLP-Dependent Oxidase, Not an Oxygenase

The PLP-dependent l-arginine hydroxylase/deaminase MppP from <i>Streptomyces wadayamensis</i> (SwMppP) is involved in the biosynthesis of l-enduracididine, a nonproteinogenic amino acid found in several nonribosomally produced peptide antibiotics. SwMppP uses only PLP and molecular oxygen to catalyze a 4-electron oxidation of l-arginine to form a mixture of 2-oxo-4­(S)-hydroxy-5-guanidinovaleric acid and 2-oxo-5-guanidinovaleric acid. Steady-state kinetics analysis in the presence and absence of catalase shows that one molecule of peroxide is formed for every molecule of dioxygen consumed in the reaction. Moreover, for each molecule of 2-oxo-4­(S)-hydroxy-5-guanidinovaleric acid produced, two molecules of dioxygen are consumed, suggesting that both the 4-hydroxy and 2-keto groups are derived from water. This was confirmed by running the reactions using either ^[18]O₂ or H₂^[18]O and analyzing the products by ESI-MS. Incorporation of ^[18]O was only observed when the reaction was performed in H₂^[18]O. Crystal structures of SwMppP with l-arginine, 2-oxo-4­(S)-hydroxy-5-guanidinovaleric acid, or 2-oxo-5-guanidinovaleric acid bound were determined at resolutions of 2.2, 1.9. and 1.8 Å, respectively. The structural data show that the N-terminal portion of the protein is disordered unless substrate or product is bound in the active site, in which case it forms a well-ordered helix that covers the catalytic center. This observation suggested that the N-terminal helix may have a role in substrate binding and/or catalysis. Our structural and kinetic characterizations of N-terminal variants show that the N-terminus is critical for catalysis. In light of this new information, we have refined our previously proposed mechanism of the SwMppP-catalyzed oxidation of l-arginine

Sbi00515, a Protein of Unknown Function from <i>Streptomyces bingchenggensis</i>, Highlights the Functional Versatility of the Acetoacetate Decarboxylase Scaffold

The acetoacetate decarboxylase-like superfamily (ADCSF) is a group of ∼4000 enzymes that, until recently, was thought to be homogeneous in terms of the reaction catalyzed. Bioinformatic analysis shows that the ADCSF consists of up to seven families that differ primarily in their active site architectures. The soil-dwelling bacterium <i>Streptomyces bingchenggensis</i> BCW-1 produces an ADCSF enzyme of unknown function that shares a low level of sequence identity (∼20%) with known acetoacetate decarboxylases (ADCs). This enzyme, Sbi00515, belongs to the MppR-like family of the ADCSF because of its similarity to the mannopeptimycin biosynthetic protein MppR from <i>Streptomyces hygroscopicus</i>. Herein, we present steady state kinetic data that show Sbi00515 does not catalyze the decarboxylation of any α- or β-keto acid tested. Rather, we show that Sbi00515 catalyzes the condensation of pyruvate with a number of aldehydes, followed by dehydration of the presumed aldol intermediate. Thus, Sbi00515 is a pyruvate aldolase-dehydratase and not an acetoacetate decarboxylase. We have also determined the X-ray crystal structures of Sbi00515 in complexes with formate and pyruvate. The structures show that the overall fold of Sbi00515 is nearly identical to those of both ADC and MppR. The pyruvate complex is trapped as the Schiff base, providing evidence that the Schiff base chemistry that drives the acetoacetate decarboxylases has been co-opted to perform a new function, and that this core chemistry may be conserved across the superfamily. The structures also suggest possible catalytic roles for several active site residues

The Aspartate-Less Receiver (ALR) Domains: Distribution, Structure and Function

<div><p>Two-component signaling systems are ubiquitous in bacteria, Archaea and plants and play important roles in sensing and responding to environmental stimuli. To propagate a signaling response the typical system employs a sensory histidine kinase that phosphorylates a Receiver (REC) domain on a conserved aspartate (Asp) residue. Although it is known that some REC domains are missing this Asp residue, it remains unclear as to how many of these divergent REC domains exist, what their functional roles are and how they are regulated in the absence of the conserved Asp. Here we have compiled all deposited REC domains missing their phosphorylatable Asp residue, renamed here as the Aspartate-Less Receiver (ALR) domains. Our data show that ALRs are surprisingly common and are enriched for when attached to more rare effector outputs. Analysis of our informatics and the available ALR atomic structures, combined with structural, biochemical and genetic data of the ALR archetype RitR from <i>Streptococcus pneumoniae</i> presented here suggest that ALRs have reorganized their active pockets to instead take on a constitutive regulatory role or accommodate input signals other than Asp phosphorylation, while largely retaining the canonical post-phosphorylation mechanisms and dimeric interface. This work defines ALRs as an atypical REC subclass and provides insights into shared mechanisms of activation between ALR and REC domains.</p></div

Structural and Functional Characterization of MppR, an Enduracididine Biosynthetic Enzyme from <i>Streptomyces hygroscopicus</i>: Functional Diversity in the Acetoacetate Decarboxylase-like Superfamily

The nonproteinogenic amino acid enduracididine is a critical component of the mannopeptimycins, cyclic glycopeptide antibiotics with activity against drug-resistant pathogens, including methicillin-resistant <i>Staphylococcus aureus</i>. Enduracididine is produced in <i>Streptomyces hygroscopicus</i> by three enzymes, MppP, MppQ, and MppR. On the basis of primary sequence analysis, MppP and MppQ are pyridoxal 5′-phosphate-dependent aminotransferases; MppR shares a low, but significant, level of sequence identity with acetoacetate decarboxylase. The exact reactions catalyzed by each enzyme and the intermediates involved in the route to enduracididine are currently unknown. Herein, we present biochemical and structural characterization of MppR that demonstrates a catalytic activity for this enzyme and provides clues about its role in enduracididine biosynthesis. Bioinformatic analysis shows that MppR belongs to a previously uncharacterized family within the acetoacetate decarboxylase-like superfamily (ADCSF) and suggests that MppR-like enzymes may catalyze reactions diverging from the well-characterized, prototypical ADCSF decarboxylase activity. MppR shares a high degree of structural similarity with acetoacetate decarboxylase, though the respective quaternary structures differ markedly and structural differences in the active site explain the observed loss of decarboxylase activity. The crystal structure of MppR in the presence of a mixture of pyruvate and 4-imidazolecarboxaldehyde shows that MppR catalyzes the aldol condensation of these compounds and subsequent dehydration. Surprisingly, the structure of MppR in the presence of “4-hydroxy-2-ketoarginine” shows the correct 4<i>R</i> enantiomer of “2-ketoenduracididine” bound to the enzyme. These data, together with bioinformatic analysis of MppR homologues, identify a novel family within the acetoacetate decarboxylase-like superfamily with divergent active site structure and, consequently, biochemical function

Crystal structure of the RitR REC domain.

<p>(<b>a</b>) Cartoon representation of RitR_ALR with helices α1- α3 and α5 colored orange, the unusual α4 helix colored green, and the β-strands colored blue. The equivalent of the phospho-modified Asp residue found in typical REC domains, RitR coordinate Asn53, is shown as ball-and-stick with blue carbon and red oxygen atoms. (<b>b</b>) Schematic view showing the pattern of RitR van der Waals interactions (yellow dotted lines) and hydrogen-bonding network (green dotted lines) in the dimer / Gate region of the structure. (<b>c</b>) Close-up of the kinked α4 helix (in green) and surrounding residues. The blue sphere is a water molecule.</p

Crystallographic data collection and model refinement statistics.

<p>^aValues in parentheses apply to the high-resolution shell indicated in the resolution row.</p><p>^bR = Σ(||Fobs|-scale*|Fcalc||) / Σ |Fobs|.</p><p>^cNumber of close interatomic contacts per 1000 atoms.</p><p>^dIsotropic equivalent B factors, including contribution from TLS refinement.</p><p>Crystallographic data collection and model refinement statistics.</p

Structure of the RitR ‘active’ pocket.

<p>(<b>a</b>) Stereoview of the electron density in the RitR active site (magenta mesh) contoured at 1.5 σ. Water molecules are shown as blue spheres. Notice the lack of a metal ion in the typical metal-binding site near Glu9. (<b>b</b>) Schematic view of the RitR REC ‘active site’ showing predicted hydrogen-bonding interactions (green dotted lines) with distances in Angstroms (Å). (<b>c</b>) Comparison of the vacuum electrostatic surface potentials of RitR_ALR, <i>left</i>, and the PhoB REC domain, <i>right</i>. The Mg²⁺ site in PhoB is indicated by a magenta sphere, which can be seen protruding slightly through the surface (denoted by the white arrow). (<b>d</b>) Comparison of the surfaces of the RitR and PhoB REC domains colored by distance from the center of mass of each protein. Not only is the electronegative environment in the metal-binding site of PhoB lost in RitR, the cleft that normally holds the metal ion (yellow region near the white arrow) is missing as well. Figure generated using PyMol (Version 1.4.1, Schrödinger, LLC).</p

SEC and NMR analysis of wild-type RitR_ALR and the RitR_ALR Leu90>Ala mutant proteins.

<p>(<b>a</b>) SEC of the proteins. V, void volume; D, dimer peak; M, monomer peak. mAU, milli Absorbance Units. (<b>b</b>) ¹H-¹⁵N HSQC spectra of the wild-type RitR_ALR (blue peaks) and Leu90>Ala mutant (green peaks). Assigned amides and NH₂ groups from Gln87 and Gln94 are also labeled. ppm, parts per million. (<b>c</b>) Heat map of chemical shift changes between the Leu90>Ala mutant and wild-type proteins. Examples of typical peaks used to calculate the degree of change between the mutant and wild-type for the heat map, and their associated coloring scheme are shown on the left. The RitR heat map structures show the proposed α4-β5 dimeric interface from two perspectives that differ by 180°. Secondary structures and residues that experienced large changes in their chemical environments when transitioning from the wild-type monomer to the mutant dimeric structure are labeled.</p

Structural and Functional Characterization of MppR, an Enduracididine Biosynthetic Enzyme from <i>Streptomyces hygroscopicus</i>: Functional Diversity in the Acetoacetate Decarboxylase-like Superfamily

The nonproteinogenic amino acid enduracididine is a critical component of the mannopeptimycins, cyclic glycopeptide antibiotics with activity against drug-resistant pathogens, including methicillin-resistant <i>Staphylococcus aureus</i>. Enduracididine is produced in <i>Streptomyces hygroscopicus</i> by three enzymes, MppP, MppQ, and MppR. On the basis of primary sequence analysis, MppP and MppQ are pyridoxal 5′-phosphate-dependent aminotransferases; MppR shares a low, but significant, level of sequence identity with acetoacetate decarboxylase. The exact reactions catalyzed by each enzyme and the intermediates involved in the route to enduracididine are currently unknown. Herein, we present biochemical and structural characterization of MppR that demonstrates a catalytic activity for this enzyme and provides clues about its role in enduracididine biosynthesis. Bioinformatic analysis shows that MppR belongs to a previously uncharacterized family within the acetoacetate decarboxylase-like superfamily (ADCSF) and suggests that MppR-like enzymes may catalyze reactions diverging from the well-characterized, prototypical ADCSF decarboxylase activity. MppR shares a high degree of structural similarity with acetoacetate decarboxylase, though the respective quaternary structures differ markedly and structural differences in the active site explain the observed loss of decarboxylase activity. The crystal structure of MppR in the presence of a mixture of pyruvate and 4-imidazolecarboxaldehyde shows that MppR catalyzes the aldol condensation of these compounds and subsequent dehydration. Surprisingly, the structure of MppR in the presence of “4-hydroxy-2-ketoarginine” shows the correct 4<i>R</i> enantiomer of “2-ketoenduracididine” bound to the enzyme. These data, together with bioinformatic analysis of MppR homologues, identify a novel family within the acetoacetate decarboxylase-like superfamily with divergent active site structure and, consequently, biochemical function