Recombinant Acinetobacter sp. N-succinylarginine dihydrolase (astB)

What is the molecular structure and catalytic mechanism of Acinetobacter sp. astB?

AstB exhibits a pseudo 5-fold symmetric alpha/beta propeller fold consisting of circularly arranged betabetaalphabeta modules that enclose the active site. Crystallographic studies have shown that AstB contains a Cys-His-Glu catalytic triad characteristic of the amidinotransferase (AT) superfamily . The enzyme catalyzes the hydrolysis of N2-succinylarginine into N2-succinylornithine, releasing ammonia and CO2 in the process .

The catalytic mechanism appears to involve two successive hydrolysis steps with ammonia release in each cycle. Crystal structures of complexes with reaction products and a C365S mutant with bound N-succinylarginine substrate suggest a mechanism similar to that proposed for arginine deiminases . Like other members of the AT superfamily, AstB possesses a flexible loop that is disordered without substrate but adopts an ordered conformation upon substrate binding, shielding the ligand from bulk solvent and controlling substrate access and product release .

What is the role of astB in the arginine succinyltransferase pathway?

AstB functions as the second enzyme in the arginine succinyltransferase pathway, which is the major pathway for arginine catabolism as a sole nitrogen source in Escherichia coli and related bacteria, including Acinetobacter species . This pathway consists of five steps, each catalyzed by a distinct enzyme:

• AstA (arginine succinyltransferase) converts arginine to N-succinylarginine

• AstB (N-succinylarginine dihydrolase) converts N-succinylarginine to N-succinylornithine

• AstC (N-succinylornithine transaminase)

• AstD (N-succinyglutamate semialdehyde dehydrogenase)

• AstE (N-succinyglutamate desuccinylase)

In pathogenic Acinetobacter species, this pathway appears to have evolved additional components, including a second ast locus (ast2) that contains astO, which enables the utilization of ornithine as a carbon source . This expanded metabolic capability may contribute to the persistence of pathogenic Acinetobacter species in specific host environments, particularly the gut .

How do mutations in the Cys-His-Glu catalytic triad affect astB function?

Mutations in the Cys-His-Glu catalytic triad of astB significantly impact its enzymatic function. Studies with similar enzymes in the amidinotransferase superfamily have shown that:

• The cysteine residue acts as the nucleophile in the catalytic mechanism

• The histidine serves as a general base

• The glutamate helps orient the histidine and stabilize reaction intermediates

In experimental studies, mutation of the catalytic cysteine to serine (C365S in E. coli AstB) has been used to trap enzyme-substrate complexes for crystallographic studies, as this mutation allows substrate binding but prevents catalysis . Similar mutations in histidine and glutamate residues would likely disrupt the proton transfer network essential for catalysis.

In a related study with A. baumannii AstA, mutations of catalytic residues (AstAL125A,H229A) were used to create a catalytically inactive enzyme while minimizing structural disruption, demonstrating the critical nature of these residues for function . These types of mutations provide valuable tools for dissecting the catalytic mechanism and for trapping reaction intermediates for structural characterization.

What is the evolutionary relationship between astB in pathogenic and non-pathogenic Acinetobacter species?

The evolutionary relationship of astB between pathogenic and non-pathogenic Acinetobacter species reveals interesting patterns related to metabolic adaptation and pathogenicity:

Phylogenetic analysis suggests that A. baumannii likely acquired the second locus (including astO) through horizontal gene transfer from a closely related species or through partial gene duplication after the divergence of pathogenic Acinetobacter clades . This additional metabolic capability provides a competitive advantage in specific host environments like the gut.

The high conservation of the ast2 locus in sequenced A. baumannii isolates (present in 83.9%, with an additional 8.5% containing a partial ast2 locus) suggests strong selective pressure to maintain this metabolic pathway .

How does astB expression influence Acinetobacter baumannii colonization in different host environments?

Research indicates that astB, as part of the arginine succinyltransferase pathway, plays a significant role in A. baumannii colonization, particularly in the gut environment:

• In mouse models, wild-type A. baumannii significantly outcompeted ΔastO mutants in gut colonization by 9 days post-infection in antibiotic-treated mice

• ΔastO mutants showed no defect in mouse models of lung infection or bloodstream infection, suggesting that the importance of this pathway is specific to the gut environment

• At 10 days post-infection, wild-type A. baumannii CFU were significantly higher than the ΔastO mutant strain in the cecum and colon, with a similar trend in the small intestine

This environmental specificity may be related to the availability of different amino acids in various host niches, with ornithine being a particular advantage in the gut. The ast2 locus appears to be highly conserved (present in 83.9% of A. baumannii isolates), suggesting strong selective pressure to maintain this metabolic capability for successful colonization .

What expression systems are optimal for producing functional recombinant Acinetobacter sp. astB?

For producing functional recombinant Acinetobacter sp. astB, E. coli-based expression systems have been successfully employed, but optimal conditions depend on specific experimental goals:

Research has shown that the timing of protein synthesis induction plays a critical role in the fate of recombinant proteins within host cells. Inducing expression during mid-log phase rather than early-log phase often results in better maintained expression levels during the late growth phase .

Comparisons between E. coli host strains (M15 and DH5α) have revealed significant differences in the expression of proteins involved in fatty acid and lipid biosynthesis pathways, with the E. coli M15 strain demonstrating superior expression characteristics for certain recombinant proteins .

What methods are recommended for determining the crystal structure of astB-substrate complexes?

Determining the crystal structure of astB-substrate complexes requires a systematic approach combining protein purification, crystallization, and structural analysis techniques:

• Protein preparation:

  • Produce highly pure (>95%) recombinant astB, preferably with minimal tags or removable tags

  • For substrate complexes, prepare catalytically inactive mutants (typically C365S for amidinotransferase family enzymes) that allow substrate binding without catalysis

• Crystallization strategy:

  • Screen initial conditions using commercial sparse matrix screens in vapor diffusion setups

  • For substrate complexes, try both co-crystallization (mixing protein with substrate before crystallization) and soaking methods (adding substrate to pre-formed crystals)

  • Optimize promising conditions by varying precipitant concentration, pH, temperature, and additives

• Data collection and processing:

  • Use synchrotron radiation sources for high-resolution data collection

  • For phase determination, molecular replacement using existing AstB structures (like E. coli AstB) is often most efficient

  • Focus refinement on the active site region to accurately model substrate interactions

Previous successful crystallization of E. coli AstB resulted in structures showing the pseudo 5-fold symmetric alpha/beta propeller fold with the Cys-His-Glu catalytic triad . These structures revealed important details about substrate binding, including the role of the flexible loop that becomes ordered upon substrate binding.

How can directed evolution be applied to enhance the catalytic efficiency of astB?

Directed evolution can be systematically applied to enhance astB's catalytic efficiency through the following methodical approach:

• Establish a screening system:

  • Develop a high-throughput screening assay that directly measures astB activity

  • Consider colorimetric detection of ammonia release or coupling product formation to a reporter system

• Create genetic diversity:

  • Use error-prone PCR to introduce random mutations across the gene

  • Implement site-saturation mutagenesis targeting the active site residues or flexible loop regions identified from structural studies

  • DNA shuffling can recombine gene fragments from different variants

• Selection strategy:

  • Transform the mutant library into an appropriate expression host lacking endogenous arginine metabolism pathways

  • Implement a metabolic complementation system where astB activity is linked to cell survival, or alternatively, a colony-based colorimetric screen

• Iterative improvement:

  • Perform multiple rounds of mutation and selection

  • Use the best performers from each round as templates for the next round

  • Consider deep sequencing between rounds to identify beneficial mutations

• Characterization of improved variants:

  • Perform detailed biochemical characterization including steady-state kinetics (kcat/KM determination)

  • Test thermal stability and substrate specificity

  • Structural analysis of improved variants can provide insights into how beneficial mutations enhance activity

This approach has been successfully applied to other members of the amidinotransferase superfamily and could be adapted specifically for astB to improve its catalytic parameters for specific experimental or biotechnological applications.

How should discrepancies in astB kinetic data between in vitro and in vivo experiments be reconciled?

Reconciling discrepancies between in vitro and in vivo astB kinetic data requires addressing multiple factors that differ between these experimental contexts:

• Physicochemical conditions:

  • In vitro assays often use optimized buffers, pH, and substrate concentrations

  • Consider performing in vitro experiments under more physiologically relevant conditions (appropriate pH, ionic strength, presence of cellular metabolites)

• Substrate accessibility:

  • In vivo, substrate concentrations may be limited by transport or competing metabolic pathways

  • Targeted metabolomics to measure intracellular N-succinylarginine concentrations can help determine if substrate availability differs from in vitro conditions

• Post-translational modifications and protein interactions:

  • Investigate modifications or protein-protein interactions present in vivo but absent in purified systems

  • Proteomics approaches comparing astB from cell lysates versus recombinant systems can identify such modifications

• Molecular crowding effects:

  • The cellular environment is highly crowded, affecting enzyme kinetics

  • Simulate crowding in vitro using crowding agents like PEG or Ficoll

• Experimental integration:

  • Construct genetic systems with varying expression levels of astB and related pathway enzymes

  • Develop computational models integrating both in vitro parameters and measured cellular conditions

Research on A. baumannii metabolism has shown that ornithine catabolism via astB is particularly important in specific environments like the gut , suggesting that the in vivo conditions in this niche may optimize enzyme function in ways that are difficult to replicate in standard in vitro assays.

What is the relationship between astB and surface-associated motility in Acinetobacter baumannii?

Studies have identified a connection between astB and surface-associated motility in Acinetobacter baumannii:

• The A. baumannii gene a1s_3129, which encodes for succinylarginine dihydrolase (astB), has been implicated in surface-associated motility

• Transposon mutants with disruptions in astB exhibited significantly reduced surface motility compared to wild-type strains

• This motility defect was demonstrated in multiple A. baumannii strains, including ATCC 17978 and 29D2

Experimental data showed that astB mutants displayed at least a 7-fold reduction in the spreading zone diameter on motility assays using 0.5% agarose plates . The mechanism linking astB function to bacterial motility remains incompletely understood but may involve:

• Changes in metabolic flux affecting energy availability for motility

• Alterations in amino acid pools affecting signaling pathways involved in motility regulation

• Potential roles in biofilm formation, which is often connected to motility phenotypes

Complementation studies would be valuable to confirm that the motility defect is specifically due to loss of astB function rather than polar effects on neighboring genes or secondary mutations.

What bioinformatic approaches are most useful for predicting astB substrate specificity?

Several complementary bioinformatic approaches are particularly effective for predicting astB substrate specificity:

• Sequence-based methods:

  • Multiple sequence alignment of astB homologs across different species identifies conserved residues in the substrate-binding pocket

  • Profile hidden Markov models (HMMs) can define substrate-binding patterns

  • Comparison between astB sequences from pathogenic Acinetobacter species (which can metabolize both arginine and ornithine) versus non-pathogenic species may reveal specificity-determining residues

• Structure-based approaches:

  • Molecular docking of potential substrates into the active site predicts binding affinity and orientation

  • Molecular dynamics simulations provide insights into the dynamic aspects of enzyme-substrate interactions

  • Analysis of available crystal structures of astB-substrate complexes identifies key binding residues

• Integrative methods:

  • Phylogenetic analysis integrated with substrate utilization data can identify evolutionary patterns linked to substrate adaptations

  • Network-based approaches examining the genomic context of astB genes provide insights into physiological substrates based on co-evolved metabolic pathways

For astB specifically, comparing sequences between the Acinetobacter baumannii complex (which encodes both astA and astO) and other Acinetobacter species (which encode only astA) has revealed important differences in substrate utilization . This comparative approach, combined with structural analysis, has helped elucidate how pathogenic Acinetobacter species evolved the ability to utilize ornithine as a carbon source through the acquisition of astO.

How can proteomics be used to study the impact of astB expression on host cell metabolism?

Proteomics provides powerful tools for investigating how astB expression impacts host cell metabolism:

• Differential expression analysis:

  • Compare protein expression profiles between wild-type cells and those expressing recombinant astB

  • Identify up- or down-regulated proteins involved in metabolic pathways

  • Studies have shown that recombinant protein production can cause significant changes in both transcriptional and translational machinery

• Temporal proteomics:

  • Analyze protein expression at different time points after astB induction

  • This approach has revealed that the timing of protein synthesis induction plays a critical role in determining the fate of recombinant proteins within the host cell

• Targeted pathway analysis:

  • Focus on specific metabolic pathways affected by astB expression

  • Previous studies have identified significant differences in the expression of proteins involved in fatty acid and lipid biosynthesis pathways between different E. coli host strains expressing recombinant proteins

• Metabolic burden assessment:

  • Quantify changes in proteins related to central carbon metabolism, amino acid biosynthesis, and energy production

  • These changes can be correlated with observed growth rate effects and recombinant protein yields

• Integration with metabolomics:

  • Combine proteomic data with metabolite profiling to create a comprehensive view of metabolic changes

  • This integrated approach can identify bottlenecks in precursor supply and energy metabolism

Proteomics studies have revealed that recombinant protein production induces significant changes in host cell metabolism, affecting growth rate, protein yield, and product formation . Understanding these effects is crucial for optimizing expression systems for astB and similar enzymes.