Cofactor F420 is a unique electron carrier in a number of microorganisms including Archaea and Mycobacteria. It has been shown that F420 has a direct and important role in archaeal energy metabolism whereas the role of F420 in mycobacterial metabolism has only begun to be uncovered in the last few years. It has been suggested that cofactor F420 has a role in the pathogenesis of M. tuberculosis, the causative agent of tuberculosis. In the absence of a commercial source for F420, M. smegmatis has previously been used to provide this cofactor for studies of the F420-dependent proteins from mycobacterial species. Three proteins have been shown to be involved in the F420 biosynthesis in Mycobacteria and three other proteins have been demonstrated to be involved in F420 metabolism. Here we report the over-expression of all of these proteins in M. smegmatis and testing of their importance for F420 production. The results indicate that co–expression of the F420 biosynthetic proteins can give rise to a much higher F420 production level. This was achieved by designing and preparing a new T7 promoter–based co-expression shuttle vector. A combination of co–expression of the F420 biosynthetic proteins and fine-tuning of the culture media has enabled us to achieve F420 production levels of up to 10 times higher compared with the wild type M. smegmatis strain. The high levels of the F420 produced in this study provide a suitable source of this cofactor for studies of F420-dependent proteins from other microorganisms and for possible biotechnological applications.
Cofactor F 420 is an electron carrier with a major role in the oxidoreductive reactions of Mycobacterium tuberculosis, the causative agent of tuberculosis. A ␥-glutamyl ligase catalyzes the final steps of the F 420 biosynthesis pathway by successive additions of L-glutamate residues to F 420 -0, producing a poly-␥-glutamate tail. The enzyme responsible for this reaction in archaea (CofE) comprises a single domain and produces F 420 -2 as the major species. The homologous M. tuberculosis enzyme, FbiB, is a two-domain protein and produces F 420 with predominantly 5-7 L-glutamate residues in the poly-␥-glutamate tail. The N-terminal domain of FbiB is homologous to CofE with an annotated ␥-glutamyl ligase activity, whereas the C-terminal domain has sequence similarity to an FMN-dependent family of nitroreductase enzymes. Here we demonstrate that full-length FbiB adds multiple L-glutamate residues to F 420 -0 in vitro to produce F 420 -5 after 24 h; communication between the two domains is critical for full ␥-glutamyl ligase activity. We also present crystal structures of the C-terminal domain of FbiB in apo-, F 420 -0-, and FMN-bound states, displaying distinct sites for F 420 -0 and FMN ligands that partially overlap. Finally, we discuss the features of a full-length structural model produced by small angle x-ray scattering and its implications for the role of N-and C-terminal domains in catalysis.The cofactor F 420 is a flavin derivative that is sporadically distributed among microorganisms, mainly archaea and actinobacteria (including mycobacteria). F 420 has been emerging as a new player in the biology of mycobacteria (1), with increasing numbers of F 420 -utilizing proteins characterized from different mycobacterial species (2-8). This cofactor has been suggested to protect Mycobacterium tuberculosis, the causative agent of tuberculosis, against oxidative and nitrosative stress during pathogenesis (9 -11). At the biochemical level, cofactor F 420 functions as a hydride transfer agent in oxidoreductive reactions with a lower redox potential than that of NAD(P) ϩ (12). The biosynthesis pathway of cofactor F 420 has been investigated in both archaeal and mycobacterial species. In the current view of the proposed pathway, the first intermediate with the complete chromophore (7, ) is produced by FO synthase (FbiC in mycobacteria (13) and CofGH in archaea (14)). A transferase enzyme (FbiA in mycobacteria (15) and CofD in archaea (16)) subsequently catalyzes the addition of a 2-phospho-L-lactate moiety to FO to produce F 420 -0 (F 420 with no poly-␥-glutamate tail). The final step of the pathway is performed by a ␥-glutamyl ligase (FbiB in mycobacteria (15) and CofE in archaea (17)) that catalyzes successive additions of L-glutamate residues to F 420 -0 (Fig. 1A).The length of the poly-␥-glutamate tail varies between archaeal and mycobacterial species; in archaea, two L-glutamate residues are seen (18), whereas in mycobacteria, up to nine residues are present (3, 19). There exists an intriguing difference between the ...
This study aims to document species presence and distribution of sea cucumbers (Echinodermata: Holothuroidea) in Malaysia. Several coral reef habitats in Peninsular Malaysia, West Malaysia and Sabah, East Malaysia were selected as study sites. In summary, the present data showed the presence of 50 species of sea cucumbers from three orders and seven genera, with 34 species require further species identification. It was found that Order Aspidochirotida in general and genus Holothuria in particular were the major species classes. The most dominant species in Malaysia was Holothuria leucospilota. As many as 37 species were found in Sabah, 21 species were recorded in Peninsular Malaysia and 10 species were present in both regions. Of 15 Actinopyga species, 14 species recorded were from Sabah. These findings may be due to the extensive distribution of coral reefs and low level of marine pollution. However, the possibility of biogeography factors within and out of the Sunda Platform area cannot be ruled out. In contrast, low level of species diversity was observed in few study sites in Peninsular Malaysia especially in Langkawi Island possibly due to anthropogenic threats. Future studies including more study sites and molecular phylogeny are to be incorporated in order to obtain better view on the presence and distribution of sea cucumbers in Malaysia. ABSTRAKKajian ini bertujuan untuk mendokumentasi kewujudan dan taburan spesis timun laut (Ekinodermata: Holothuroidea) di Malaysia. Beberapa habitat batu karang di Semenanjung Malaysia, Malaysia Barat dan Sabah, Malaysia Timur telah dipilih sebagai kawasan kajian. Sebagai rumusan, data terkini menunjukkan kehadiran 50 spesis timun laut dari tiga order dan tujuh genus di seluruh Malaysia, di mana 34 spesis memerlukan pengesahan lanjut. Order Aspidokirotida amnya dan genus Holothuria khususnya menunjukkan kehadiran spesis yang tertinggi. Holothuria leucospilota secara bandingannya adalah spesis paling dominan di Malaysia. Sebanyak 37 spesis telah dijumpai di Sabah, 21 spesis direkodkan di Semenanjung Malaysia dan 10 spesis ditemui di kedua-dua kawasan tersebut. Daripada 15 spesis Actinopyga yang direkodkan, 14 spesis adalah dari Sabah. Penemuan ini mungkin disebabkan taburan batu karang yang luas dan tahap pencemaran laut yang rendah. Walau bagaimanapun, kebarangkalian faktor-faktor biogeografi di dalam atau di luar Pentas Sunda perlu diambilkira. Sebaliknya, kepelbagaian spesis di beberapa kawasan di Semenanjung Malaysia didapati rendah terutamanya di Pulau Langkawi kemungkinan disebabkan oleh ancaman manusia. Kajian lanjut melibatkan lebih banyak kawasan kajian dan filogeni molekul akan dijalankan pada masa hadapan untuk mendapatkan gambaran lebih jelas mengenai kehadiran dan taburan timun laut di Malaysia.
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