Evolutionary and functional insights into Leishmania META1: evidence for lateral gene transfer and a role for META1 in secretion.

BACKGROUND: Leishmania META1 has for long been a candidate molecule for involvement in virulence: META1 transcript and protein are up-regulated in metacyclic Leishmania. Yet, how META1 contributes to virulence remains unclear. We sought insights into the possible functions of META1 by studying its evolutionary origins. RESULTS: Using multiple criteria including sequence similarity, nucleotide composition, phylogenetic analysis and selection pressure on gene sequence, we present evidence that META1 originated in trypanosomatids as a result of a lateral gene transfer of a bacterial heat-inducible protein, HslJ. Furthermore, within the Leishmania genome, META1 sequence is under negative selection pressure against change/substitution. Using homology modeling of Leishmania META1 based on solved NMR structure of HslJ, we show that META1 and HslJ share a similar structural fold. The best hit for other proteins with similar fold is MxiM, a protein involved in the type III secretion system in Shigella. The striking structural similarity shared by META1, HslJ and MxiM suggests a possibility of shared functions. Upon structural superposition with MxiM, we have observed a putative hydrophobic cavity in META1. Mutagenesis of select hydrophobic residues in this cavity affects the secretion of the secreted acid phosphatase (SAP), indicating META1's involvement in secretory processes in Leishmania. CONCLUSIONS: Overall, this work uses an evolutionary biology approach, 3D-modeling and site-directed mutagenesis to arrive at new insights into functions of Leishmania META1

A type III PKS makes the DIFference

Differentiation-inducing factor is a modified polyketide natural product involved in the differentiation of Dictyostelium discoideum cells. A new study shows that a type III polyketide synthase existing in an unusual association with type I fatty acid synthase domains is responsible for biosynthesis of this signaling compound

Review The fi del ity of the trans la tion of the ge netic code *.

Aminoacyl-tRNA syn the tas es play a cen tral role in main tain ing ac cu racy dur ing the trans la tion of the ge netic code. To achieve this chal leng ing task they have to dis crim i-nate against amino ac ids that are very closely re lated not only in struc ture but also in chem i cal na ture. A ‘dou ble-sieve ’ ed it ing model was pro posed in the late sev en ties to ex plain how two closely re lated amino ac ids may be dis crim i nated. How ever, a clear un der stand ing of this mech a nism re quired struc tural in for ma tion on syn the tas es that are faced with such a prob lem of amino acid dis crim i na tion. The first struc tural ba sis for the ed it ing model came re cently from the crys tal struc ture of isoleucyl-tRNA synthetase, a class I synthetase, which has to dis crim i nate against valine. The struc-ture showed the pres ence of two cat a lytic sites in the same en zyme, one for ac ti va tion, a coarse sieve which binds both isoleucine and valine, and an other for ed it ing, a fine sieve which binds only valine and re jects isoleucine. An other struc ture of the en zyme in com plex with tRNA showed that the tRNA is re spon si ble for the translocation of the misactivated amino-acid sub strate from the cat a lytic site to the ed it ing site. These stud ies were mainly fo cused on class I syn the tas es and the sit u a tion was not clear about how class II en zymes dis crim i nate against sim i lar amino ac ids. The re cent struc tural and en zy matic stud ies on threonyl-tRNA synthetase, a class II en zyme, re-veal how this chal leng ing task is achieved by us ing a unique zinc ion in the ac tive site as well as by em ploy ing a sep a rate do main for spe cific ed it ing ac tiv ity. These stud ie

Versatility of polyketide synthases in generating metabolic diversity

Polyketide synthases (PKSs) form a large family of multifunctional proteins involved in the biosynthesis of diverse classes of natural products. Architecturally at least three different types of PKSs have been discovered in the microbial world and recent years have revealed tremendous versatility of PKSs, both in terms of their structural and functional organization and in their ability to produce compounds other than typical secondary metabolites. Mycobacterium tuberculosis exploits polyketide biosynthetic enzymes to synthesize complex lipids, many of which are essential for its survival. The functional significance of the large repertoire of PKSs in Dictyostelium discoideum, perhaps in producing developmental regulating factors, is emerging. Recently determined structures of fatty acid synthases (FASs) and PKSs now provide an opportunity to delineate the mechanistic and structural basis of polyketide biosynthetic machinery

Fatty acyl-AMP ligases and polyketide synthases are unique enzymes of lipid biosynthetic machinery in Mycobacterium tuberculosis

The cell envelope of Mycobacterium tuberculosis (Mtb) possesses a repertoire of unusual lipids that are believed to play an important role in pathogenesis. In this review, we specifically focus on computational, biochemical and structural studies in lipid biosynthesis that have established functional role of polyketide synthases (PKSs) and fatty acyl-AMP ligases (FAALs). Mechanistic and structural studies with FAALs suggest that this group of proteins may have evolved from omnipresent fatty acyl-CoA ligases (FACLs). FAALs activate fatty acids as acyl-adenylates and transfer them on to the PKSs which then produce unusual acyl chains that are the components of mycobacterial lipids. FAALs are a newly discovered family of enzymes; whereas involvement of PKSs in lipid metabolism was not known prior to their discovery in Mtb. Since Mtb genome contains multiple homologs of FAALs and PKSs and owing to the conserved reaction mechanism and overlapping substrate specificity; there is tempting opportunity to develop 'systemic drugs' against these enzymes as anti-tuberculosis agents

A D-amino acid editing module coupled to the translational apparatus in archaea

We report the crystal structure of an archaea-specific editing domain of threonyl-tRNA synthetase that reveals a marked structural similarity to D-amino acid deacylases found in eubacteria and eukaryotes. The domain can bind D-amino acids despite a low sequence identity to other D-amino acid deacylases. These results together indicate the presence of these deacylases in all three kingdoms of life. This underlines an important role they may have played in enforcing homochirality during translation

Exploring the limits of sequence and structure in a variant βγ-crystallin domain of the protein absent in melanoma-1 (AIM1)

βγ-Crystallins belong to a superfamily of proteins in prokaryotes and eukaryotes that are based on duplications of a characteristic, highly conserved Greek key motif. Most members of the superfamily in vertebrates are structural proteins of the eye lens that contain four motifs arranged as two structural domains. Absent in melanoma 1 (AIM1), an unusual member of the superfamily whose expression is associated with suppression of malignancy in melanoma, contains 12 βγ-crystallin motifs in six domains. Some of these motifs diverge considerably from the canonical motif sequence. AIM1g1, the first βγ-crystallin domain of AIM1, is the most variant of βγ-crystallin domains currently known. In order to understand the limits of sequence variation on the structure, we report the crystal structure of AIM1g1 at 1.9 Å resolution. Despite having changes in key residues, the domain retains the overall βγ-crystallin fold. The domain also contains an unusual extended surface loop that significantly alters the shape of the domain and its charge profile. This structure illustrates the resilience of the βγ fold to considerable sequence changes and its remarkable ability to adapt for novel functions

A cell wall degrading esterase of Xanthomonas oryzae requires a unique substrate recognition module for pathogenesis on rice

Xanthomonas oryzae pv oryzae (Xoo) causes bacterial blight, a serious disease of rice (Oryza sativa). LipA is a secretory virulence factor of Xoo, implicated in degradation of rice cell walls and the concomitant elicitation of innate immune responses, such as callose deposition and programmed cell death. Here, we present the high-resolution structural characterization of LipA that reveals an all-helical ligand binding module as a distinct functional attachment to the canonical hydrolase catalytic domain. We demonstrate that the enzyme binds to a glycoside ligand through a rigid pocket comprising distinct carbohydrate-specific and acyl chain recognition sites where the catalytic triad is situated 15 Å from the anchored carbohydrate. Point mutations disrupting the carbohydrate anchor site or blocking the pocket, even at a considerable distance from the enzyme active site, can abrogate in planta LipA function, exemplified by loss of both virulence and the ability to elicit host defense responses. A high conservation of the module across genus Xanthomonas emphasizes the significance of this unique plant cell wall-degrading function for this important group of plant pathogenic bacteria. A comparison with the related structural families illustrates how a typical lipase is recruited to act on plant cell walls to promote virulence, thus providing a remarkable example of the emergence of novel functions around existing scaffolds for increased proficiency of pathogenesis during pathogen-plant coevolution