Structures and function of the amino acid polymerase cyanophycin synthetase.

Cyanophycin is a natural biopolymer produced by a wide range of bacteria, consisting of a chain of poly-L-Asp residues with L-Arg residues attached to the ß-carboxylate sidechains by isopeptide bonds. Cyanophycin is synthesized from ATP, aspartic acid and arginine by a homooligomeric enzyme called cyanophycin synthetase (CphA1). CphA1 has domains that are homologous to glutathione synthetases and muramyl ligases, but no other structural information has been available. Here, we present cryo-electron microscopy and X-ray crystallography structures of cyanophycin synthetases from three different bacteria, including cocomplex structures of CphA1 with ATP and cyanophycin polymer analogs at 2.6 Å resolution. These structures reveal two distinct tetrameric architectures, show the configuration of active sites and polymer-binding regions, indicate dynamic conformational changes and afford insight into catalytic mechanism. Accompanying biochemical interrogation of substrate binding sites, catalytic centers and oligomerization interfaces combine with the structures to provide a holistic understanding of cyanophycin biosynthesis.

Piecing together nonribosomal peptide synthesis.

Nonribosomal peptide synthetases (NRPSs) produce peptide products with wide-ranging biological activities. NRPSs are macromolecular machines with modular assembly-line logic, a complex catalytic cycle, moving parts and multiple active sites. They are organized into repeating sets of domains, called modules. Each module contains all functionality to introduce a building block into the growing peptide, many also perform cosynthetic tailoring. Structures of individual domains have provided insights into their catalytic mechanisms, but with one exception, larger NRPS proteins were refractory to structure determination. Recently, structure determination succeeded for four multi-domain NRPS proteins: an alternative formylating initiation and two termination modules as well as a large cross-module construct. This review highlights how these data, together with novel didomain structures, contribute to a holistic view of the architecture, domain-domain interactions and conformational changes in NRPS megaenzymes.

A mutation in an exoglucanase of Xanthomonas oryzae pv. oryzae, which confers an endo mode of activity, affects bacterial virulence, but not the induction of immune responses, in rice.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, a serious disease of rice. Xoo secretes a repertoire of cell wall-degrading enzymes, including cellulases, xylanases and pectinases, to degrade various polysaccharide components of the rice cell wall. A secreted Xoo cellulase, CbsA, is not only a key virulence factor of Xoo, but is also a potent inducer of innate immune responses of rice. In this study, we solved the crystal structure of the catalytic domain of the CbsA protein to a resolution of 1.86 Å. The core structure of CbsA shows a central distorted TIM barrel made up of eight ß strands with N- and C-terminal loops enclosing the active site, which is a characteristic structural feature of an exoglucanase. The aspartic acid at the 131st position of CbsA was predicted to be important for catalysis and was therefore mutated to alanine to study its role in the catalysis and biological functions of CbsA. Intriguingly, the D131A CbsA mutant protein displayed the enzymatic activity of a typical endoglucanase. D131A CbsA was as proficient as wild-type (Wt) CbsA in inducing rice immune responses, but was deficient in virulence-promoting activity. This indicates that the specific exoglucanase activity of the Wt CbsA protein is required for this protein to promote the growth of Xoo in rice.

X-Ray Crystallography and Electron Microscopy of Cross- and Multi-Module Nonribosomal Peptide Synthetase Proteins Reveal a Flexible Architecture.

Nonribosomal peptide synthetases (NRPS) are macromolecular machines that produce peptides with diverse activities. Structural information exists for domains, didomains, and even modules, but little is known about higher-order organization. We performed a multi-technique study on constructs from the dimodular NRPS DhbF. We determined a crystal structure of a cross-module construct including the adenylation (A) and peptidyl carrier protein (PCP) domains from module 1 and the condensation domain from module 2, complexed with an adenosine-vinylsulfonamide inhibitor and an MbtH-like protein (MLP). The action of the inhibitor and the role of the MLP were investigated using adenylation reactions and isothermal titration calorimetry. In the structure, the PCP and A domains adopt a novel conformation, and noncovalent, cross-module interactions are limited. We calculated envelopes of dimodular DhbF using negative-stain electron microscopy. The data show large conformational variability between modules. Together, our results suggest that NRPSs lack a uniform, rigid supermodular architecture.

Structural insights into the regulation of NADPH binding to reductase domains of nonribosomal peptide synthetases: A concerted loop movement model.

The termination module of nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) offloads the final product as an acid (occasionally also accompanied by cyclization) upon hydrolysis by employing thioesterase domains (TE-domains). Reductase domains (R-domains) of short-chain dehydrogenase/reductase (SDR) family offer an alternative offloading mechanism by reducing 4'-phosphopantetheine (4'-PPant) arm-tethered peptidyl chain, a thioester, to an aldehyde or an alcohol. Recent studies have highlighted their functional importance, for instance in the glycopeptidolipid (GPL) biosynthesis of Mycobacterium smegmatis, where the resulting alcoholic group is the site for subsequent modifications such as glycosylations. The mechanistic understanding of how these R-domains function in the context of multi-modular NRPS and PKS is poorly understood. In this study, conformational differences in functionally important loops, not reported previously, were identified in a new crystal form of R-domain which may be relevant to functioning in the context of assembly-line NRPS and PKS enzymology. Here, we propose a concerted loop movement model that allows gating of cofactor binding to these enzymes, enabling the release of the final product only after the substrate has reached the active site during biosynthesis, and therefore distinct from a canonical single domain SDR family of enzymes.

Unsaturated Lipid Assimilation by Mycobacteria Requires Auxiliary cis-trans Enoyl CoA Isomerase.

Mycobacterium tuberculosis (Mtb) can survive in hypoxic necrotic tissue by assimilating energy from host-derived fatty acids. While the expanded repertoire of ß-oxidation auxiliary enzymes is considered crucial for Mtb adaptability, delineating their functional relevance has been challenging. Here, we show that the Mtb fatty acid degradation (FadAB) complex cannot selectively break down cis fatty acyl substrates. We demonstrate that the stereoselective binding of fatty acyl substrates in the Mtb FadB pocket is due to the steric hindrance from Phe287 residue. By developing a functional screen, we classify the family of Mtb Ech proteins as monofunctional or bifunctional enzymes, three of which complement the FadAB complex to degrade cis fatty acids. Crystal structure determination of two cis-trans enoyl coenzyme A (CoA) isomerases reveals distinct placement of active-site residue in Ech enzymes. Our studies thus reveal versatility of Mtb lipid-remodeling enzymes and identify an essential role of stand-alone cis-trans enoyl CoA isomerases in mycobacterial biology.

Mutations in the Predicted Active Site of Xanthomonas oryzae pv. oryzae XopQ Differentially Affect Virulence, Suppression of Host Innate Immunity, and Induction of the HR in a Nonhost Plant.

Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, secretes a number of effectors through a type 3 secretion system. One of these effectors, called XopQ, is required for virulence and suppression of rice innate immune responses induced by the plant cell-wall-degrading enzyme lipase/esterase A (LipA). Bioinformatic analysis suggested that XopQ is homologous to inosine-uridine nucleoside hydrolases (NH). A structural model of XopQ with the protozoan Crithidia fasciculata purine NH suggested that D116 and Y279 are potential active site residues. X. oryzae pv. oryzae xopQ mutants (xopQ-/pHM1::xopQD116A and xopQ-/pHM1::xopQY279A) show reduced virulence on rice compared with xopQ-/pHM1::xopQ. The two predicted XopQ active site mutants (xopQ-/pHM1::xopQD116A and xopQ-/pHM1::xopQY279A) exhibit a reduced hypersensitive response (HR) on Nicotiana benthamiana, a nonhost. However, Arabidopsis lines expressing either xopQ or xopQY279A are equally proficient at suppression of LipA-induced callose deposition. Purified XopQ does not show NH activity on standard nucleoside substrates but exhibits ribose hydrolase activity on the nucleoside substrate analogue 4-nitrophenyl ß-D-ribofuranoside. The D116A and Y279A mutations cause a reduction in biochemical activity. These results indicate that mutations in the predicted active site of XopQ affect virulence and induction of the HR but do not affect suppression of innate immunity.

Delineating the reaction mechanism of reductase domains of Nonribosomal Peptide Synthetases from mycobacteria.

Substrate binding to enzymes often follows a precise order where catalysis is accomplished through programmed conformational changes. Short-chain dehydrogenase/reductase (SDR) enzymes follow sequential order 'bi-bi' reaction kinetics. The mechanistic study of a SDR homolog, reductase (R) domain, from multifunctional enzymes, e.g. Nonribosomal Peptide Synthetases (NRPSs) and Polyketide Synthases (PKSs) has revealed that it reductively releases 4'-phosphopantetheinyl arm-tethered peptidyl product. We report that the R-domains of NRPSs from Mycobacterium tuberculosis (RNRP) and Mycobacterium smegmatis (RGPL) do not strictly adhere to the obligatory mode of catalysis performed by SDRs, but instead can carry out reductive catalysis of substrate following random bi-bi reaction mechanism as deciphered by NMR and SAXS studies. The crucial conformational change associated with NADPH binding necessary to achieve catalytically competent conformation is also delineated by SAXS studies. Using ITC, we have demonstrated that mutation of catalytic tyrosine to phenylalanine in R-domains results in 3-4-fold decrease in affinity for NADPH and attribute this phenomenon to loss of the noncovalent cation-π interactions present between the tyrosine and nicotinamide ring. We propose that the adaptation to an alternative theme of bi-bi catalytic mechanism enables the R-domains to process the substrates transferred by upstream domains and maintain assembly-line enzymology.

Crystallization and preliminary crystallographic studies of CbsA, a secretory exoglucanase from Xanthomonas oryzae pv. oryzae.

The bacterial pathogen Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. The secreted exoglucanase CbsA is an important virulence factor of this pathogen. It belongs to the glycosyl hydrolase 6 family of proteins based on the carbohydrate-active enzyme (CAZY) classification. In this study, CbsA has been overexpressed, purified and crystallized. The crystal diffracted to a resolution of 1.86 Šand belonged to space group P2(1)2(1)2(1). It contained one monomer per asymmetric unit, with a solvent content of 45.8%.

Nonprocessive [2 + 2]e- off-loading reductase domains from mycobacterial nonribosomal peptide synthetases.

In mycobacteria, polyketide synthases and nonribosomal peptide synthetases (NRPSs) produce complex lipidic metabolites by using a thio-template mechanism of catalysis. In this study, we demonstrate that off-loading reductase (R) domain of mycobacterial NRPSs performs two consecutive [2 + 2]e(-) reductions to release thioester-bound lipopeptides as corresponding alcohols, using a nonprocessive mechanism of catalysis. The first crystal structure of an R domain from Mycobacterium tuberculosis NRPS provides strong support to this mechanistic model and suggests that the displacement of intermediate would be required for cofactor recycling. We show that 4e(-) reductases produce alcohols through a committed aldehyde intermediate, and the reduction of this intermediate is at least 10 times more efficient than the thioester-substrate. Structural and biochemical studies also provide evidence for the conformational changes associated with the reductive cycle. Further, we show that the large substrate-binding pocket with a hydrophobic platform accounts for the remarkable substrate promiscuity of these domains. Our studies present an elegant example of the recruitment of a canonical short-chain dehydrogenase/reductase family member as an off-loading domain in the context of assembly-line enzymology.