Allelic association with ankylosing spondylitis fails to correlate with human leukocyte antigen B27 homodimer formation

Expression of human leukocyte antigen (HLA)-B27 is strongly associated with predisposition toward ankylosing spondylitis (AS) and other spondyloarthropathies. However, the exact involvement of HLA-B27 in disease initiation and progression remains unclear. The homodimer theory, which proposes that HLA-B27 heavy chains aberrantly form homodimers, is a central hypothesis that attempts to explain the role of HLA-B27 in disease pathogenesis. Here, we examined the ability of the eight most prevalent HLA-B27 allotypes (HLA-B*27:02 to HLA-B*27:09) to form homodimers. We observed that HLA-B*27:03, a disease-associated HLA-B27 subtype, showed a significantly reduced ability to form homodimers compared with all other allotypes, including the non-disease-associated/protective allotypes HLA-B*27:06 and HLA-B*27:09. We used X-ray crystallography and site-directed mutagenesis to unravel the molecular and structural mechanisms in HLA-B*27:03 that are responsible for its compromised ability to form homodimers. We show that polymorphism at position 59, which differentiates HLA-B*27:03 from all other allotypes, is responsible for its compromised ability to form homodimers. Indeed, histidine 59 in HLA-B*27:03 leads to a series of local conformational changes that act in concert to reduce the accessibility of the nearby cysteine 67, an essential amino acid residue for the formation of HLA-B27 homodimers. Considered together, the ability of both protective and disease-associated HLA-B27 allotypes to form homodimers and the failure of HLA-B*27:03 to form homodimers challenge the role of HLA-B27 homodimers in AS pathoetiology. Rather, this work implicates other features, such as peptide binding and antigen presentation, as pivotal mechanisms for disease pathogenesis

Cytochrome P450-mediated hydroxylation is required for polyketide macrolactonization in stambomycin biosynthesis

International audienceMany polyketide antibiotics contain macrolactones that arise from polyketide synthase chain release via thioesterase (TE) domain-catalyzed macrolactonization. The hydroxyl groups utilized in such macrolactonization reactions typically derive from reduction of beta-ketothioester intermediates in polyketide chain assembly. The stambomycins are a group of novel macrolide antibiotics with promising anticancer activity that we recently discovered via rational activation of a silent polyketide biosynthetic gene cluster in Streptomyces ambofaciens. Here we report that the hydroxyl group utilized for formation of the macrolactone in the stambomycins is derived from cytochrome P450-catalyzed hydroxylation of the polyketide chain rather than keto reduction during chain assembly. This is a novel mechanism for macrolactone formation in polyketide antibiotic biosynthesis

Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F-420-0 in Mycobacteria

F420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo-, substrate-, and product-bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic precomplex of this enzyme and demonstrate that dehydro-F420-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance.IMPORTANCE Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F420, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F420 is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F420, determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F420