Dissection of the EntF condensation domain boundary and active site residues in nonribosomal peptide synthesis

Nonribosomal peptide synthetases (NRPSs) make many natural products of clinical importance, but a deeper understanding of the protein domains that compose NRPS assembly lines is required before these megasynthetases can be effectively engineered to produce novel drugs. The N-terminal amide bond-forming condensation (C) domain of the enterobactin NRPS EntF was excised from the multidomain synthetase using endpoints determined from sequence alignments and secondary structure predictions. The isolated domain was well-folded when compared by circular dichroism to the vibriobactin NRPS VibH, a naturally free-standing C domain. The EntF domain was also fully functional in an assay based on a synthetic small-molecule substrate, seryl N-acetylcysteamine. Active site mutants of the EntF C domain were surprisingly inactive in vitro as compared to their VibH counterparts, yet maintained the overall domain structure. An in vivo assay was developed in the context of the full-length EntF protein to more sensitively probe the activity level of the C domain mutants, and this supported strong effects for the active site mutations. The crucial role of histidine-138 was confirmed by assay of the full-length protein in vitro. These results suggest a strong resemblance of catalysis by the EntF C domain to chloramphenicol acetyltransferase, including an active site organized by an arginine-aspartate salt bridge, a key histidine acting as a general base, and an asparagine instead of a serine stabilizing the proposed tetrahedral intermediate by hydrogen bonding. The precise definition of a functional C domain excised from a NRPS should aid efforts at swapping NRPS domains between assembly lines.     

Vibriobactin biosynthesis in Vibrio cholerae: VibH is an amide synthase homologous to nonribosomal peptide synthetase condensation domains

The Vibrio cholerae siderophore vibriobactin is biosynthesized from three molecules of 2,3-dihydroxybenzoate (DHB), two molecules of L-threonine, and one of norspermidine. Of the four genes positively implicated in vibriobactin biosynthesis, we have here expressed, purified, and assayed the products of three: vibE, vibB, and vibH. All three are homologous to nonribosomal peptide synthetase (NRPS) domains: VibE is a 2,3-dihydroxybenzoate-adenosyl monophosphate ligase, VibB is a bifunctional isochorismate lyase-aryl carrier protein (ArCP), and VibH is a novel amide synthase that represents a free-standing condensation (C) domain. VibE and VibB are homologous to EntE and EntB from Escherichia coli enterobactin synthetase; VibE activates DHB as the acyl adenylate and then transfers it to the free thiol of the phosphopantetheine arm of VibB's ArCP domain. VibH then condenses this DHB thioester (the donor) with the small molecule norspermidine (the acceptor), forming N(1)-(2, 3-dihydroxybenzoyl)norspermidine (DHB-NSPD) with a k(cat) of 600 min(-1) and a K(m) for acyl-VibB of 0.88 microM and for norspermidine of 1.5 mM. Exclusive monoacylation of a primary amine of norspermidine was observed. VibH also tolerates DHB-acylated EntB and 1,7-diaminoheptane, octylamine, and hexylamine as substrates, albeit at lowered catalytic efficiencies. DHB-NSPD possesses one of three acylations required for mature vibriobactin, and its formation confirms VibH's role in vibriobactin biosynthesis. VibH is a unique NRPS condensation domain that acts upon an upstream carrier-protein-bound donor and a downstream amine, turning over a soluble amide product, in contrast to an archetypal NRPS-embedded C domain that condenses two carrier protein thioesters.     

Dimeric structure of the six-domain VibF subunit of vibriobactin synthetase: mutant domain activity regain and ultracentrifugation studies

Nonribosomal peptide synthetases (NRPS), fatty acid synthases (FAS), and polyketide sythases (PKS) are multimodular enzymatic assembly lines utilized in natural product biosynthesis. Previous data on FAS and PKS subunits have indicated that they are homodimers and that some of their catalytic functions can work in trans. When NRPS assembly lines have been probed for comparable formation of stable oligomers, no evidence had been forthcoming that species other than monomer forms were active. In this work we focus on the six-domain (Cy1-Cy2-A-C1-PCP-C2) enzyme VibF from the vibriobactin synthetase assembly line, which contains three other proteins, VibB, VibE, and VibH, that--when purified and mixed with VibF and the substrates ATP, threonine, 2,3-dihydroxybenzoate (DHB), and norspermidine--produce the iron chelator vibriobactin. Using a deletion of the Cy1 domain and separate inactivating mutations in the Cy2, A, PCP, and C2 domains of VibF, we report regain of catalytic activity upon mutant protein mixing that argues for heterodimer formation, stable for hundreds to thousands of catalytic cycles, with acyl chain processing and transfer around blocked domains. Ultracentrifugation data likewise confirm a dimeric structure for VibF and establish that domains within NRPS dimeric modules can act on acyl chains in trans. The results described here are the first indication for an NRPS subunit that homodimerization can occur and that there is a continuum of functional oligomerization states between monomers and dimers in nonribosomal peptide synthetases.     

Carrier protein recognition in siderophore-producing nonribosomal peptide synthetases

Nonribosomal peptide synthetases (NRPSs) use phosphopantetheine (pPant) bearing carrier proteins to chaperone activated aminoacyl and peptidyl intermediates to the various enzymes that effect peptide synthesis. Using components from siderophore NRPSs that synthesize vibriobactin, enterobactin, yersiniabactin, pyochelin, and anguibactin, we examined the nature of the interaction of such cofactor-carrier proteins with acyl-activating adenylation (A) domains. While VibE, EntE, and PchD were all able to utilize "carrier protein-free" pPant derivatives, the pattern of usage indicated diversity in the binding mechanism, and even the best substrates were down at least 3 log units relative to the native cofactor-carrier protein. When tested with four noncognate carrier proteins, EntE and VibE differed both in the range of substrate utilization efficiency and in the distribution of the efficiencies across this range. Correlating sequence alignments to kinetic efficiency allowed for the construction of eight point mutants of VibE's worst substrate, HMWP2 ArCP, to the corresponding residue in its native VibB. Mutants S49D and H66E combined to increase activity 6.2-fold and had similar enhancing effects on the downstream condensation domain VibH, indicating that the two NRPS enzymes share carrier protein recognition determinants. Similar mutations of HMWP2 ArCP toward EntB had little effect on EntE, suggesting that the position of recognition determinants varies across NRPS systems.     

Unraveling terminal C-domain-mediated condensation in fungal biosynthesis of imidazoindolone metabolites

The fungal peptidyl alkaloids of the tryptoquialanine and fumiquinazoline families are nonribosomally assembled by annulation of the indole side chain of fumiquinazoline F (FQF) with an alaninyl or aminoisobutyryl unit by monomodular NRPS enzymes containing adenylation, thiolation, and condensation (A-T-C) domains. The Af12060 and Af12050 enzyme pair from Aspergillus fumigatus thereby converts FQF to FQA, while the homologous TqaH and TqaB enzyme pair from Penicillium aethiopicum makes the 2'-epi diastereomer of FQA, differing only in the stereochemistry of one of the C-N bonds formed in the annulation with l-Ala. To evaluate the basis for this stereochemical control, we have mixed and matched the flavoprotein oxygenases Af12060 and TqaH with the A-T-C modular enzymes Af12050 and TqaB to show that the NRPS enzymes control the stereochemical outcome. The terminal 50 kDa condensation domains of Af12050 and TqaB are solely responsible for the stereochemical control as shown both by making chimeric (e.g., A-T-C* and A*-T*-C) forms of these monomodular NRPS enzymes and by expression, purification, and assay of the excised C-domains. The Af12050 and TqaB condensation domains are thus a paired set of diastereospecific annulation catalysts that act on the fumiquinazoline F scaffold.     

Serratia marcescens gene required for surfactant serrawettin W1 production encodes putative aminolipid synthetase belonging to nonribosomal peptide synthetase family

Serrawettin W1 produced by Serratia marcescens is a surface active exolipid having various functions supporting behaviors of bacteria on surface environments. Through the genetic analyses of serrawettin-less mutants of S. marcescens 274, the swrW gene encoding putative serrawettin W1 synthetase was identified. Homology analysis of the putative SwrW demonstrated the presence of condensation, adenylation, thiolation, and thioesterase domains which are characteristic for nonribosomal peptide synthetase (NRPS). NRPSs have been known as multi-modular enzymes. Linear alignment of these modules specifying respective amino acids will enable peptide bond formation resulting in a specific amino acid sequence. Putative SwrW was uni-modular NRPS specifying only L-serine. Possible steps in this simple unimodular NRPS for biosynthesis of serrawettin W1 [ cyclo-(D-3-hydroxydecanoyl-L-seryl) (2) ] were predicted by referring to the ingenious enzymatic activity of gramicidin S synthetase (multi-modular NRPS) of Brevibacillus brevis.     

Chain initiation in the leinamycin-producing hybrid nonribosomal peptide/polyketide synthetase from Streptomyces atroolivaceus S-140. Discrete, monofunctional adenylation enzyme and peptidyl carrier protein that directly load D-alanine

Nonribosomal peptide natural products are biosynthesized from amino acid precursors by nonribosomal peptide synthetases (NRPSs), which are organized into modules. For a typical NRPS initiation module, an adenylation (A) domain activates an amino acid and installs it onto a peptidyl carrier protein (PCP) domain as a thioester; an elongation module, which has a condensation (C) domain located between every consecutive pair of A and PCP domains, catalyzes the formation of the peptide bond between the upstream aminoacyl/peptidyl-S-PCP and the free amino group of the downstream aminoacyl-S-PCP. D-amino acid constituents in peptide natural products usually arise from the L-enantiomers through the action of integral epimerization (E) domains of an NRPS. The biosynthetic gene cluster for leinamycin, a hybrid nonribosomal peptide/polyketide containing a D-alanine moiety, does not encode a typical NRPS initiation module with the expected A-PCP-E domains; instead, it has only an A protein (LnmQ) and a PCP (LnmP), both of which are encoded by separate genes. Here we show the results of biochemical experiments as follows: (i) we demonstrate that LnmQ directly activates D-alanine as D-alaninyl-AMP and installs it onto LnmP to generate a D-alaninyl-S-PCP intermediate; (ii) we confirm that aminoacylation of LnmP by LnmQ in trans is the result of specific communication between the separate A and PCP proteins; and (iii) we reveal that leinamycin production can be improved by supplementation of exogenous D-alanine in the fermentation broth of Streptomyces atroolivaceous S-140. These findings unveil an unprecedented NRPS initiation module structure that is characterized by a discrete D-alanine-specific A protein and a PCP.     

Substrate selection of adenylation domains for nonribosomal peptide synthetase (NRPS) in bacillamide C biosynthesis by marine <Emphasis Type="Italic">Bacillus atrophaeus</Emphasis> C89

Nonribosomal peptide synthetases (NRPSs) are multi-modular enzymes involved in the biosynthesis of natural products. Bacillamide C was synthesized by Bacillus atrophaeus C89. A nonribosomal peptide synthetase (NRPS) cluster found in the genome of B. atrophaeus C89 was hypothesized to be responsible for the biosynthesis of bacillamide C using alanine and cysteine as substrates. Here, the structure analysis of adenylation domains based on homologous proteins with known crystal structures indicated locations of the substrate-binding pockets. Molecular docking suggested alanine and cysteine as the potential substrates for the two adenylation domains in the NRPS cluster. Furthermore, biochemical characterization of the purified recombinant adenylation domains proved that alanine and cysteine were the optimum substrates for the two adenylation domains. The results provided the in vitro evidence for the hypothesis that the two adenylation domains in the NRPS of B. atrophaeus C89 preferentially select alanine and cysteine, respectively, as a substrate to synthesize bacillamide C. Furthermore, this study on substrates selectivity of adenylation domains provided basis for rational design of bacillamide analogs.     

Catalytic mapping of the vibriobactin biosynthetic enzyme VibF.

The iron-chelating catechol siderophore vibriobactin of the pathogenic Vibrio cholerae is assembled by a four-subunit, ten-domain nonribosomal peptide synthetase system, VibE, VibB, VibH, and VibF, using 2,3-dihydroxybenzoate and L-threonine as precursors to two (dihydroxyphenyl)methyloxazolinyl groups in amide linkage on a norspermidine scaffold. We have utilized site-specific and domain-deletion mutagenesis to map the heterocyclization and primary and secondary amine acylation activities of the six-domain (Cy1-Cy2-A-C1-PCP-C2) VibF subunit. We have found that Cy2 is capable of and limited to the condensation (amide bond formation) step of the three-step heterocyclization process, while Cy1 is capable of and limited to the final processing (cyclization/dehydration) steps to the completed heterocycle. Additionally, we have observed that the C2 domain functions in both N(9) (primary amine) acylation and N(5) (secondary amine) acylation of the (dihydroxybenzoyl)norspermidine substrate, leaving no catalytic role for the C1 domain, a conclusion confirmed with the formation of vibriobactin in a C1-deficient system. Thus VibF is an NRPS with two domains, Cy1 and Cy2, that perform a function otherwise performed by one and with one domain, C2, that performs a function otherwise performed by two. While C2 appeared to tolerate uncyclized threonine in place of the usual heterocycle in primary amine acylation, it refused this replacement in the corresponding donor substrate in secondary amine acylation.     

Deep sequencing of non-ribosomal peptide synthetases and polyketide synthases from the microbiomes of Australian marine sponges

The biosynthesis of non-ribosomal peptide and polyketide natural products is facilitated by multimodular enzymes that contain domains responsible for the sequential condensation of amino and carboxylic subunits. These conserved domains provide molecular targets for the discovery of natural products from microbial metagenomes. This study demonstrates the application of tag-encoded FLX amplicon pyrosequencing (TEFAP) targeting non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes as a method for determining the identity and diversity of natural product biosynthesis genes. To validate this approach, we assessed the diversity of NRPS and PKS genes within the microbiomes of six Australian marine sponge species using both TEFAP and metagenomic whole-genome shotgun sequencing approaches. The TEFAP approach identified 100 novel ketosynthase (KS) domain sequences and 400 novel condensation domain sequences within the microbiomes of the six sponges. The diversity of KS domains within the microbiome of a single sponge species Scopalina sp. exceeded that of any previously surveyed marine sponge. Furthermore, this study represented the first to target the condensation domain from NRPS biosynthesis and resulted in the identification of a novel condensation domain lineage. This study highlights the untapped potential of Australian marine sponges for the isolation of novel bioactive natural products. Furthermore, this study demonstrates that TEFAP approaches can be applied to functional genes, involved in natural product biosynthesis, as a tool to aid natural product discovery. It is envisaged that this approach will be used across multiple environments, offering an insight into the biological processes that influence the production of secondary metabolites.     

The 1.8 A crystal structure of PA2412, an MbtH-like protein from the pyoverdine cluster of Pseudomonas aeruginosa

Many bacteria use nonribosomal peptide synthetase (NRPS) proteins to produce peptide antibiotics and siderophores. The catalytic domains of the NRPS proteins are usually linked in large multidomain proteins. Often, additional proteins are coexpressed with NRPS proteins that modify the NRPS peptide products, ensure the availability of substrate building blocks, or play a role in the import or export of the NRPS product. Many NRPS clusters include a small protein of approximately 80 amino acids with homology to the MbtH protein of mycobactin synthesis in Mycobacteria tuberculosis; no function has been assigned to these proteins. Pseudomonas aeruginosa utilizes an NRPS cluster to synthesize the siderophore pyoverdine. The pyoverdine peptide contains a dihydroxyquinoline-based chromophore, as well as two formyl-N-hydroxyornithine residues, which are involved in iron binding. The pyoverdine cluster contains four modular NRPS enzymes and 10-15 additional proteins that are essential for pyoverdine production. Coexpressed with the pyoverdine synthetic enzymes is a 72-amino acid MbtH-like family member designated PA2412. We have determined the three-dimensional structure of the PA2412 protein and describe here the structure and the location of conserved regions. Additionally, we have further analyzed a deletion mutant of the PA2412 protein for growth and pyoverdine production. Our results demonstrate that PA2412 is necessary for the production or secretion of pyoverdine at normal levels. The PA2412 deletion strain is able to use exogenously produced pyoverdine, showing that there is no defect in the uptake or utilization of the iron-pyoverdine complex.     

肽基载体蛋白结构功能的研究进展

肽基载体蛋白(peptidyl carrier protein,PCP)是非核糖体肽合成酶(non-ribosomal peptide synthetase,NRPS)的核心结构域。根据NRPS的装配机制,每个模块都至少包含一个PCP,PCP对于非核糖体肽合成中氨基酸残基及多肽在不同催化结构域中的传递起着重要作用,并为氨基酸残基和多肽向模块内其他修饰酶的转移提供一个平台。本文主要对PCP的结构功能、与其他催化结构域的相互作用及重组模块活性降低的问题等方面进行了综述,期望为重组NRPS模块的构建提供理论依据。     

Reconstitution and characterization of the Vibrio cholerae vibriobactin synthetase from VibB, VibE, VibF, and VibH

Vibriobactin [N(1)-(2,3-dihydroxybenzoyl)-N(5),N(9)-bis[2-(2, 3-dihydroxyphenyl)-5-methyloxazolinyl-4-carboxamido]norspermidine] , is an iron chelator from the cholera-causing bacterium Vibrio cholerae. The six-domain, 270 kDa nonribosomal peptide synthetase (NRPS) VibF, a component of vibriobactin synthetase, has been heterologously expressed in Escherichia coli and purified. VibF has an unusual NRPS domain organization: cyclization-cyclization-adenylation-condensation-peptidyl carrier protein-condensation (Cy(1)-Cy(2)-A-C(1)-PCP-C(2)). VibF activates and covalently loads its PCP with L-threonine, and together with vibriobactin synthetase proteins VibE (adenylation) and VibB (aryl carrier protein) condenses and heterocyclizes 2, 3-dihydroxybenzoyl-VibB with L-Thr to 2-dihydroxyphenyl-5-methyloxazolinyl-4-carboxy-VibF in the first demonstration of oxazoline formation by an NRPS cyclization domain. This enzyme-bound aryl oxazoline can be transferred by VibF to various amine acceptors but most efficiently to N(1)-(2, 3-dihydroxybenzoyl)norspermidine (k(cat) = 122 min(-1), K(m) = 1.7 microM), the product of 2,3-dihydroxybenzoyl-VibB, norspermidine, and VibH. This diacylated product undergoes a second aryl oxazoline acylation on its remaining secondary amine, also catalyzed by VibF, to yield vibriobactin. Vibriobactin biosynthesis in vitro has thus been accomplished from four proteins, VibE, VibB, VibF, and VibH, with the substrates 2,3-dihydroxybenzoic acid, L-Thr, norspermidine, and ATP. Vibriobactin synthetase is an unusual NRPS in that all intermediates are not covalently tethered as PCP thioesters and in that it represents an NRPS pathway with two branch points.     

Structure of a Eukaryotic Nonribosomal Peptide Synthetase Adenylation Domain That Activates a Large Hydroxamate Amino Acid in Siderophore Biosynthesis

Nonribosomal peptide synthetases (NRPSs) are large, multidomain proteins that are involved in the biosynthesis of an array of secondary metabolites. We report the structure of the third adenylation domain from the siderophore-synthesizing NRPS, SidN, from the endophytic fungus Neotyphodium lolii. This is the first structure of a eukaryotic NRPS domain, and it reveals a large binding pocket required to accommodate the unusual amino acid substrate, N^δ-cis-anhydromevalonyl-N^δ-hydroxy-l-ornithine (cis-AMHO). The specific activation of cis-AMHO was confirmed biochemically, and an AMHO moiety was unambiguously identified as a component of the fungal siderophore using mass spectroscopy. The protein structure shows that the substrate binding pocket is defined by 17 amino acid residues, in contrast to both prokaryotic adenylation domains and to previous predictions based on modeling. Existing substrate prediction methods for NRPS adenylation domains fail for domains from eukaryotes due to the divergence of their signature sequences from those of prokaryotes. Thus, this new structure will provide a basis for improving prediction methods for eukaryotic NRPS enzymes that play important and diverse roles in the biology of fungi.     

Non-ribosomal peptide synthetases: Identifying the cryptic gene clusters and decoding the natural product

Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are the major multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically important antibiotics and siderophores. Each of the multiple modules of an NRPS activates a different amino or aryl acid, followed by their condensation to synthesize a linear or cyclic natural product. The studies on NRPS domains, the knowledge of their gene cluster architecture and tailoring enzymes have helped in the in silico genetic screening of the ever-expanding sequenced microbial genomic data for the identification of novel NRPS/PKS clusters and thus deciphering novel non-ribosomal peptides (NRPs). Adenylation domain is an integral part of the NRPSs and is the substrate selecting unit for the final assembled NRP. In some cases, it also requires a small protein, the MbtH homolog, for its optimum activity. The presence of putative adenylation domain and MbtH homologs in a sequenced genome can help identify the novel secondary metabolite producers. The role of the adenylation domain in the NRPS gene clusters and its characterization as a tool for the discovery of novel cryptic NRPS gene clusters are discussed.     

The Landscape of Recombination Events That Create Nonribosomal Peptide Diversity

Nonribosomal peptides (NRP) are crucial molecular mediators in microbial ecology and provide indispensable drugs. Nevertheless, the evolution of the flexible biosynthetic machineries that correlates with the stunning structural diversity of NRPs is poorly understood. Here, we show that recombination is a key driver in the evolution of bacterial NRP synthetase (NRPS) genes across distant bacterial phyla, which has guided structural diversification in a plethora of NRP families by extensive mixing and matching of biosynthesis genes. The systematic dissection of a large number of individual recombination events did not only unveil a striking plurality in the nature and origin of the exchange units but allowed the deduction of overarching principles that enable the efficient exchange of adenylation (A) domain substrates while keeping the functionality of the dynamic multienzyme complexes. In the majority of cases, recombination events have targeted variable portions of the A_core domains, yet domain interfaces and the flexible A_sub domain remained untapped. Our results strongly contradict the widespread assumption that adenylation and condensation (C) domains coevolve and significantly challenge the attributed role of C domains as stringent selectivity filter during NRP synthesis. Moreover, they teach valuable lessons on the choice of natural exchange units in the evolution of NRPS diversity, which may guide future engineering approaches.     

Roles of type II thioesterases and their application for secondary metabolite yield improvement

A large number of antibiotics and other industrially important microbial secondary metabolites are synthesized by polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). These multienzymatic complexes provide an enormous flexibility in formation of diverse chemical structures from simple substrates, such as carboxylic acids and amino acids. Modular PKSs and NRPSs, often referred to as megasynthases, have brought about a special interest due to the colinearity between enzymatic domains in the proteins working as an “assembly line” and the chain elongation and modification steps. Extensive efforts toward modified compound biosynthesis by changing organization of PKS and NRPS domains in a combinatorial manner laid good grounds for rational design of new structures and their controllable biosynthesis as proposed by the synthetic biology approach. Despite undeniable progress made in this field, the yield of such “unnatural” natural products is often not satisfactory. Here, we focus on type II thioesterases (TEIIs)—discrete hydrolytic enzymes often encoded within PKS and NRPS gene clusters which can be used to enhance product yield. We review diverse roles of TEIIs (removal of aberrant residues blocking the megasynthase, participation in substrate selection, intermediate, and product release) and discuss their application in new biosynthetic systems utilizing PKS and NRPS parts.     

Thioesterase portability and peptidyl carrier protein swapping in yersiniabactin synthetase from Yersinia pestis

Multimodular enzymes, including polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), and mixed PKS/NRPS systems, contain functional domains with similar functions. Domain swapping and module fusion are potential powerful strategies for creating hybrid enzymes to synthesize modified natural products. To explore these strategies, yersiniabactin (Ybt) synthetase containing two subunits, HMWP2 [two NRPS modules (N-terminus-ArCP-Cy1-A-PCP1 and Cy2-PCP2-C-terminus)] and HMWP1 [one PKS (N-terminus-KS-AT-MT1-KR-ACP) one NRPS module (Cy3-MT2-PCP3-TE-C-terminus)], was used as a model system to study peptidyl carrier protein (PCP) domain swapping, thioesterase (TE) portability, and module-module fusion. The PCP1 domain of the N-terminal NRPS module of HMWP2 was swapped with either PCP2 or PCP3. The fusion proteins were 3-8-fold less active than the wild-type protein. The swapping of PCP2 of HMWP2 abolished the heterocyclization activity of the Cy2 domain while retaining its condensation function. When the two PCPs of HMWP2 were swapped by PCP3TE, it created two active fusion proteins: one or two NRPS modules fused to the TE domain. The internal TE domain of the two fusion proteins catalyzed the hydrolysis of enzyme-bound intermediates HPT-S-PCP3 to form HPT-COOH and HPTT-S-PCP3 to form HPTT-COOH. The TE activity was eliminated by the S2980A point mutation at its active site. Therefore, the three PCPs of the Ybt synthetase were swappable, and its lone TE domain was portable. The reasons for the observed low activities of the fusion proteins and lessons for protein engineering in generating novel modular enzymes were discussed.     

Function of MbtH homologs in nonribosomal peptide biosynthesis and applications in secondary metabolite discovery

Mycobacterium tuberculosis encodes mycobactin, a peptide siderophore that is biosynthesized by a nonribosomal peptide synthetase (NRPS) mechanism. Within the mycobactin biosynthetic gene cluster is a gene that encodes a 71-amino-acid protein MbtH. Many other NRPS gene clusters harbor mbtH homologs, and recent genetic, biochemical, and structural studies have begun to shed light on the function(s) of these proteins. In some cases, MbtH-like proteins are required for biosynthesis of their cognate peptides, and non-cognate MbtH-like proteins have been shown to be partially complementary. Biochemical studies revealed that certain MbtH-like proteins participate in tight binding to NRPS proteins containing adenylation (A) domains where they stimulate adenylation reactions. Expression of MbtH-like proteins is important for a number of applications, including optimal production of native and genetically engineered secondary metabolites produced by mechanisms that employ NRPS enzymes. They also may serve as beacons to identify gifted actinomycetes and possibly other bacteria that encode multiple functional NRPS pathways for discovery of novel secondary metabolites by genome mining.