Recombinant Enterobacteria phage T7 N-acetylmuramoyl-L-alanine amidase (3.5)

What is the structural composition of T7 N-acetylmuramoyl-L-alanine amidase?

T7 lysozyme (also known as T7 N-acetylmuramoyl-L-alanine amidase) is a bifunctional protein found exclusively in T7 bacteriophage. Its structure has been determined by X-ray crystallography and refined at 2.2 Å resolution. The protein folds into an alpha-helical structure with a zinc-binding site essential for its catalytic activity .

The enzyme contains specific domains responsible for its dual functionality:

• A catalytic domain containing zinc-binding motifs

• An RNA polymerase interaction region

The structure, cataloged as PDB 1lba, reveals that T7 lysozyme belongs to the CATH domain 3.40.80.10 . The protein contains one zinc ion as a cofactor, which is critical for its amidase function. The zinc-binding amino acids are conserved between this enzyme and other related amidases .

What are the dual biological functions of T7 N-acetylmuramoyl-L-alanine amidase?

T7 N-acetylmuramoyl-L-alanine amidase performs two distinct biological functions that are crucial for T7 phage development:

• Amidase Activity: It cleaves the amide bond between N-acetylmuramic acid and L-alanine in the bacterial cell wall peptidoglycan layer. This enzymatic activity contributes to bacterial cell lysis during the late stages of phage infection .

• Transcriptional Regulation: It acts as an inhibitor of T7 RNA polymerase, providing a feedback mechanism that shuts off late transcription during infection and stimulates DNA replication .

This dual functionality sets T7 lysozyme apart from other phage lysozymes, as it plays both structural and regulatory roles in the phage life cycle. The transcriptional inhibition function appears to be particularly important for controlling the transition between transcription and DNA replication phases during phage infection .

How does T7 N-acetylmuramoyl-L-alanine amidase differ from other lysozymes?

T7 lysozyme differs from well-studied egg-white and phage T4 lysozymes in two significant ways:

• Mechanism of Lysis: T7 lysozyme cuts the amide bond between N-acetylmuramic acid and L-alanine, while conventional lysozymes cleave the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan layer .

• Regulatory Function: Unlike other lysozymes, T7 lysozyme interacts with T7 RNA polymerase to regulate viral gene expression .

These differences reflect the specialized evolutionary adaptations of T7 bacteriophage. The unique bond specificity of T7 lysozyme classifies it as an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) rather than a conventional lysozyme, despite its traditional naming as "lysozyme" in the literature .

What is the catalytic mechanism of T7 N-acetylmuramoyl-L-alanine amidase?

The catalytic mechanism of T7 N-acetylmuramoyl-L-alanine amidase follows a pathway similar to other zinc proteases, specifically carboxypeptidase A. The proposed mechanism involves:

• A positively charged lysine forming a bond to the carbonyl group of the amide link, polarizing it

• Nucleophilic attack by a zinc-bound hydroxide activated by a negatively charged tyrosine

• Bond cleavage and donation of a proton to the leaving amino group

The enzyme is zinc-dependent, with the metal ion playing a crucial role in activating a water molecule for nucleophilic attack on the amide bond. The minimum peptidoglycan fragment that can be hydrolyzed by the enzyme is MurNAc-tripeptide .

Specific amino acid residues essential for catalytic activity have been identified through mutational studies, including the zinc-binding amino acids that are conserved between T7 amidase and related enzymes .

What substrate specificity does T7 N-acetylmuramoyl-L-alanine amidase exhibit?

T7 N-acetylmuramoyl-L-alanine amidase exhibits specific substrate requirements:

• It hydrolyzes the amide bond between N-acetylmuramic acid (MurNAc) and L-alanine in bacterial peptidoglycan

• The minimum peptidoglycan fragment that can be hydrolyzed is MurNAc-tripeptide

• Despite its peptidoglycan-cleaving activity, the enzyme alone does not possess direct bacteriolytic activity

This substrate specificity is important for researchers designing activity assays or considering applications of the enzyme. The requirement for a MurNAc-tripeptide minimum substrate suggests that the enzyme recognizes structural features beyond the immediate cleavage site, which may have implications for its specificity toward different bacterial peptidoglycans.

What role does zinc play in T7 N-acetylmuramoyl-L-alanine amidase function?

Zinc is essential for the catalytic activity of T7 N-acetylmuramoyl-L-alanine amidase:

• The enzyme is classified as a zinc-dependent amidase (EC 3.5.1.28)

• One zinc ion serves as a cofactor in the active site

• Zinc-binding amino acids are highly conserved between T7 amidase and related enzymes

• The metal ion is required for activating a water molecule for nucleophilic attack during catalysis

Experimental evidence demonstrates that removal of zinc or mutation of zinc-binding residues results in loss of enzymatic activity. This zinc dependency is a common feature shared with human PGRP-L and other related amidases, suggesting an evolutionarily conserved catalytic mechanism .

What expression systems are optimal for producing recombinant T7 N-acetylmuramoyl-L-alanine amidase?

Successful expression of recombinant T7 N-acetylmuramoyl-L-alanine amidase requires careful consideration of expression systems. Based on research findings:

• Escherichia coli Expression Systems: E. coli Rosetta strains have been successfully used for expression, particularly when producing fusion proteins with the enzyme .

• Vector Systems: T7Select phage-based expression systems have been employed for producing the enzyme and fusion constructs .

• Expression Tags: Green fluorescent protein (GFP) tags have been used to monitor expression levels in infected E. coli cells. The addition of an N-terminal GFP tag allowed for efficient tracking of expression without significantly compromising enzymatic activity .

When designing expression strategies, researchers should consider:

• Codon optimization for the host organism

• Inclusion of appropriate zinc in growth media

• Temperature and induction conditions to minimize inclusion body formation

• Careful design of fusion constructs to maintain proper folding and activity

How can researchers design activity assays for T7 N-acetylmuramoyl-L-alanine amidase?

Designing effective activity assays for T7 N-acetylmuramoyl-L-alanine amidase requires consideration of its specific catalytic properties:

• Substrate Selection: The minimum substrate is MurNAc-tripeptide, but larger peptidoglycan fragments or purified bacterial cell walls can also be used .

• Detection Methods:

  • Monitoring the release of L-alanine using colorimetric or fluorometric assays

  • Analyzing reaction products by HPLC or mass spectrometry

  • Using labeled substrates to track bond cleavage

• Assay Conditions:

  • Buffer composition: typically includes Tris-HCl (pH 7.0-8.0)

  • Zinc supplementation: 0.01-0.1 mM ZnCl₂

  • Temperature: optimal activity at 25-37°C

  • Presence of reducing agents (e.g., DTT) to maintain cysteine residues

• Controls:

  • Negative controls with heat-inactivated enzyme

  • Positive controls with commercial amidases

  • Metal chelation controls (EDTA) to demonstrate zinc dependency

A typical activity assay protocol would involve incubating the enzyme with peptidoglycan substrate, stopping the reaction at defined timepoints, and quantifying either substrate consumption or product formation using appropriate analytical methods.

What techniques can be used to study the interaction between T7 N-acetylmuramoyl-L-alanine amidase and T7 RNA polymerase?

The interaction between T7 N-acetylmuramoyl-L-alanine amidase and T7 RNA polymerase represents a key regulatory mechanism in T7 phage biology. Researchers can employ several techniques to study this interaction:

• Biochemical Approaches:

  • Co-immunoprecipitation assays

  • Pull-down assays using tagged proteins

  • Size exclusion chromatography to detect complex formation

  • Surface plasmon resonance to measure binding kinetics

• Functional Assays:

  • In vitro transcription assays measuring inhibition of T7 RNA polymerase activity

  • Electrophoretic mobility shift assays to detect protein-protein complexes

  • Fluorescence resonance energy transfer (FRET) using labeled proteins

• Structural Studies:

  • X-ray crystallography of the complex

  • Cryo-electron microscopy

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Mutagenesis Approaches:

  • Alanine scanning mutagenesis to identify critical residues

  • Domain deletion studies to map interaction regions

  • Chimeric protein construction to verify domain functions

These approaches have helped establish that T7 lysozyme serves as an inhibitor of T7 RNA polymerase, providing a feedback mechanism that regulates the transition from transcription to DNA replication during phage infection .

How can T7 N-acetylmuramoyl-L-alanine amidase be engineered for enhanced activity or novel functions?

Engineering T7 N-acetylmuramoyl-L-alanine amidase for enhanced or novel functions requires sophisticated protein engineering approaches:

• Site-Directed Mutagenesis:

  • Targeting zinc-binding residues to modify metal coordination

  • Altering substrate-binding residues to change specificity

  • Modifying the RNA polymerase interaction domain to adjust regulatory function

• Domain Fusion Approaches:

  • Creating fusion proteins with additional antimicrobial domains

  • Combining with cell-penetrating peptides for enhanced delivery

  • Developing reporter fusions for tracking enzyme localization

• Directed Evolution Strategies:

  • Using fluorescence-activated droplet sorting (FADS) for high-throughput screening

  • Employing phage display to select variants with desired properties

  • Applying DNA shuffling to combine beneficial mutations

Recent research has demonstrated the feasibility of engineering T7 bacteriophages to express fusion proteins containing amidase domains along with antimicrobial peptides. For example, researchers have successfully integrated apidaecin-related peptide sequences into the T7Select phage genome, resulting in the expression of these peptides in infected host cells .

What is the comparative structure-function relationship between T7 N-acetylmuramoyl-L-alanine amidase and human PGRP-L?

The structural and functional relationship between T7 N-acetylmuramoyl-L-alanine amidase and human Peptidoglycan Recognition Protein-L (PGRP-L) reveals fascinating evolutionary conservation:

• Functional Similarities:

  • Both are Zn²⁺-dependent N-acetylmuramoyl-L-alanine amidases (EC 3.5.1.28)

  • Both hydrolyze the amide bond between MurNAc and L-Ala in bacterial peptidoglycan

  • Both require zinc for catalytic activity

• Structural Conservation:

  • Zinc-binding amino acids are conserved between PGRP-L and T7 amidase

  • The C-terminal region of PGRP-L is homologous to bacteriophage and bacterial amidases

  • This C-terminal region is sufficient for amidase activity, although with reduced efficiency in deletion mutants

• Key Differences:

  • Cys-419 is required for PGRP-L amidase activity but is not conserved in T7 amidase

  • Three amino acids needed for T7 amidase activity are not required for PGRP-L activity

  • PGRP-L lacks the RNA polymerase inhibitory function of T7 amidase

This comparative analysis suggests that while the core amidase function has been conserved across prokaryotes, insects, and mammals, specific adaptations have occurred to meet the specialized needs of each organism. The conservation of this enzymatic function across diverse organisms points to its fundamental importance in bacterial cell wall recognition and processing .

How can T7 N-acetylmuramoyl-L-alanine amidase be integrated into engineered bacteriophage systems?

Integration of T7 N-acetylmuramoyl-L-alanine amidase into engineered bacteriophage systems represents an emerging area of research with applications in synthetic biology and antimicrobial development:

• Genome Integration Strategies:

  • Insertion of modified amidase genes into the phage genome

  • Positioning downstream of gene product 10 (viral capsid) to leverage late-stage expression

  • Engineering fusion constructs with reporter proteins (like GFP) to monitor expression

• Functional Enhancement Approaches:

  • Combining amidase with antimicrobial peptides for synergistic activity

  • Modifying substrate specificity to target resistant bacterial strains

  • Engineering regulatory domains to control lysis timing

• Expression Optimization:

  • Codon optimization for target bacterial hosts

  • Engineering stronger promoters for higher expression levels

  • Modifying translation initiation regions to enhance protein synthesis

Recent research has demonstrated the feasibility of engineering T7 bacteriophages to express not only the native amidase but also additional antimicrobial components. Researchers have successfully integrated apidaecin-related peptide sequences and green fluorescent protein (GFP) tags into the T7Select phage genome, enabling expression in infected host cells .

Key considerations when designing such systems include:

• Ensuring proper folding and activity of the engineered proteins

• Maintaining phage viability and replication efficiency

• Optimizing expression levels to achieve the desired biological effect

• Addressing potential resistance mechanisms in target bacteria

What are common challenges in expressing active recombinant T7 N-acetylmuramoyl-L-alanine amidase?

Researchers frequently encounter several challenges when expressing active recombinant T7 N-acetylmuramoyl-L-alanine amidase:

• Expression Level Issues:

  • Low expression levels, particularly with certain fusion constructs

  • Expression below detection limits for some engineered variants

  • Variable expression depending on host strain and conditions

• Protein Folding Challenges:

  • Improper folding leading to inclusion body formation

  • Difficulties maintaining proper zinc coordination

  • Structural integrity issues with fusion proteins

• Host Cell Interactions:

  • Potential toxicity to expression hosts due to cell wall degradation

  • Growth inhibition of host cells

  • Selection for resistant bacterial subpopulations

Researchers have reported that when certain sequences, such as Api802, Api806, and Api810, were integrated into the T7Select phage genome, expression was below detection limits. Furthermore, the effect on the growth of potentially phage-resistant E. coli Rosetta strains was not observed for some constructs, suggesting challenges in achieving functional expression .

What strategies can optimize the enzymatic activity of recombinant T7 N-acetylmuramoyl-L-alanine amidase?

Optimizing the enzymatic activity of recombinant T7 N-acetylmuramoyl-L-alanine amidase requires attention to several key factors:

• Zinc Coordination:

  • Supplementing expression media and purification buffers with zinc

  • Avoiding strong chelating agents during purification

  • Characterizing the zinc:protein stoichiometry to ensure proper metalation

• Protein Stability Enhancement:

  • Identifying and modifying unstable regions through rational design

  • Adding stabilizing mutations based on computational predictions

  • Optimizing buffer conditions for long-term storage

• Fusion Partner Selection:

  • Designing optimal linker sequences for fusion proteins

  • Selecting fusion partners that enhance solubility without compromising activity

  • Considering removable fusion tags with appropriate protease sites

• Expression System Optimization:

  • Testing different E. coli strains, particularly those optimized for disulfide bond formation

  • Exploring lower temperature expression to improve folding

  • Using specialized media formulations with controlled zinc availability

The optimization process typically requires iterative testing and characterization of multiple variants. For instance, when GFP-tagged apidaecin analogs were produced in E. coli Rosetta cells infected with engineered T7Select phages, reasonable quantities of the fusion proteins were obtained, demonstrating the potential for successful expression optimization .

How can researchers troubleshoot DNA modifications in T7 phage expressing recombinant N-acetylmuramoyl-L-alanine amidase?

Troubleshooting DNA modifications in engineered T7 phage systems requires systematic investigation of several potential issues:

• Genome Stability Assessment:

  • Regular sequencing of phage genomes to detect spontaneous mutations

  • PCR verification of inserted sequences after multiple phage passages

  • Restriction enzyme analysis to confirm preserved genetic organization

• Expression Verification Approaches:

  • Using reporter fusion proteins (like GFP) to monitor expression levels

  • Western blot analysis with specific antibodies

  • Activity assays to confirm functional enzyme production

• Host-Phage Interaction Analysis:

  • Monitoring for the emergence of phage-resistant bacterial populations

  • Characterizing changes in host cell morphology during infection

  • Assessing phage burst size and infection kinetics with modified phages

• Integration Site Optimization:

  • Testing alternative genome positions for inserting recombinant sequences

  • Evaluating the impact of insert size on phage packaging efficiency

  • Designing inserts with compatible genetic elements (promoters, terminators)

Researchers have employed these approaches to troubleshoot T7 bacteriophages engineered to express antimicrobial peptides and fusion proteins. For example, when monitoring the expression of GFP-apidaecin analogs in infected E. coli Rosetta cells, researchers could detect the production of fusion proteins, although some constructs showed expression below detection limits, highlighting the need for optimization strategies .

What are the prospects for using modified T7 N-acetylmuramoyl-L-alanine amidase in reducing dsRNA production during in vitro transcription?

Recent research has explored the potential of modified T7 RNA polymerase systems to reduce double-stranded RNA (dsRNA) production during in vitro transcription, which has implications for the regulatory function of T7 N-acetylmuramoyl-L-alanine amidase:

• Engineering T7 RNA Polymerase Variants:

  • Researchers have developed T7 RNA polymerase variants (including Mut1, Mut7, Mut11, Mut14, and Mut17) with reduced dsRNA production

  • These variants show dsRNA reduction ranging from 0.18% to 1.80% compared to wild-type polymerase

  • The combinatorial mutant Mut17 (A70Q/F162S/K180E) exhibited significant reduction in dsRNA production

• Mechanistic Understanding:

  • Research has identified a correlation between dsRNA production and the activities of T7 RNA polymerase in terminal transferase and RNA-dependent RNA polymerase (RDRP) functions

  • Terminal transferase activity appears to play a critical role in dsRNA generation

• Implications for T7 Amidase Function:

  • Since T7 N-acetylmuramoyl-L-alanine amidase naturally regulates T7 RNA polymerase activity, understanding these interactions could inform new approaches to controlling transcription fidelity

  • Engineering both the polymerase and its amidase regulator could provide novel tools for synthetic biology applications

This research direction has significant implications for mRNA synthesis applications, as reduced dsRNA production leads to lower immune stress responses in cells treated with the resulting mRNA products .

How can T7 N-acetylmuramoyl-L-alanine amidase contribute to understanding phage-host interactions during different growth phases?

T7 N-acetylmuramoyl-L-alanine amidase plays a crucial role in phage-host interactions, with particular relevance to different bacterial growth phases:

• Stationary Phase Adaptation:

  • Research has identified that T7 development in E. coli requires inhibition of different forms of bacterial RNA polymerase

  • While the protein Gp2 inhibits the housekeeping form (Eσ70), another protein, Gp5.7, inhibits the stationary phase form (EσS)

  • This demonstrates how phages have evolved distinct mechanisms to facilitate development in bacteria in different growth phases

• Regulatory Network Analysis:

  • The accumulation of guanosine pentaphosphate [(p)ppGpp] during T7 development influences bacterial transcription machinery

  • T7 has evolved specific mechanisms to inhibit the bacterial transcription machinery that predominates in different growth conditions

• Research Applications:

  • Studying these interactions provides insights into bacterial stress responses

  • Understanding growth phase-specific phage-host interactions can inform the development of more effective phage therapy approaches

  • The mechanisms may reveal new targets for antimicrobial development

This research area demonstrates how T7 bacteriophage employs multiple strategies to manage host cell resources across different growth conditions, with T7 N-acetylmuramoyl-L-alanine amidase being part of a sophisticated regulatory network .

What are the emerging applications of T7 N-acetylmuramoyl-L-alanine amidase in engineered phage therapy approaches?

Engineered phage therapy represents an exciting frontier where T7 N-acetylmuramoyl-L-alanine amidase plays a crucial role:

• Synergistic Antimicrobial Approaches:

  • Researchers have genetically modified T7Select phages targeting Escherichia coli by integrating DNA sequences derived from antimicrobial peptides

  • This approach aims to trigger peptide expression by the bacterial host early in the infectious cycle

  • The goal is to create synergistic effects between phage lysis mechanisms and antimicrobial peptide activity

• Addressing Host Range Limitations:

  • Engineering phages to express antimicrobial peptides could alleviate host range limitations

  • Local production of peptide antibiotics at the infection site may address phage-resistant bacterial subpopulations

  • This approach shows promise for treating poly-bacterial infections

• Technical Advances and Challenges:

  • Successful integration of antimicrobial peptide genes downstream of gene product 10 (viral capsid)

  • Optimization of expression levels remains challenging

  • Further research is needed to achieve the expected synergistic effects between phages and antimicrobial peptides

These emerging applications highlight the potential of T7 N-acetylmuramoyl-L-alanine amidase and engineered T7 phages in next-generation antimicrobial strategies .