Diversity and novel mutations in membrane transporters of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), encodes a family of membrane proteins belonging to Resistance-Nodulation-Cell Division (RND) permeases also called multidrug resistance pumps. Mycobacterial membrane protein Large (MmpL) transporters represent a subclass of RND transporters known to participate in exporting of lipid components across the cell envelope. These proteins perform an essential role in MTB survival; however, there are no data regarding mutations in MmpL, polyketide synthase (PKS) and acyl-CoA dehydrogenase FadE proteins from Khyber Pakhtunkhwa, Pakistan. This study aimed to screen mutations in transmembrane transporter proteins including MmpL, PKS and Fad through whole-genome sequencing (WGS) in local isolates of Khyber Pakhtunkhwa province, Pakistan. Fourteen samples were collected from TB patients and drug susceptibility testing was performed. However, only three samples were completely sequenced. Moreover, 209 whole-genome sequences of the same geography were also retrieved from NCBI GenBank to analyze the diversity of mutations in MmpL, PKS and Fad proteins. Among the 212 WGS (Accession ID: PRJNA629298, PRJNA629388, and ERR2510337-ERR2510345, ERR2510546-ERR2510645), numerous mutations in Fad (n = 756), PKS (n = 479), and MmpL (n = 306) have been detected. Some novel mutations were also detected in MmpL, PKS and acyl-CoA dehydrogenase Fad. Novel mutations including Asn576Ser in MmpL8, Val943Gly in MmpL9 and Asn145Asp have been detected in MmpL3. The presence of a large number of mutations in the MTB membrane may have functional consequences on proteins. However, further experimental studies are needed to elucidate the variants' effect on MmpL, PKS and FadE functions.

Beta-oxidation of 5-hydroxydecanoate, a putative blocker of mitochondrial ATP-sensitive potassium channels.

5-Hydroxydecanoate (5-HD) inhibits ischaemic and pharmacological preconditioning of the heart. Since 5-HD is thought to inhibit specifically the putative mitochondrial ATP-sensitive K+ (KATP) channel, this channel has been inferred to be a mediator of preconditioning. However, it has recently been shown that 5-HD is a substrate for acyl-CoA synthetase, the mitochondrial enzyme which 'activates' fatty acids. Here, we tested whether activated 5-HD, 5-hydroxydecanoyl-CoA (5-HD-CoA), is a substrate for medium-chain acyl-CoA dehydrogenase (MCAD), the committed step of the mitochondrial beta-oxidation pathway. Using a molecular model, we predicted that the hydroxyl group on the acyl tail of 5-HD-CoA would not sterically hinder the active site of MCAD. Indeed, we found that 5-HD-CoA was a substrate for purified human liver MCAD with a Km of 12.8 +/- 0.6 microM and a kcat of 14.1 s-1. For comparison, with decanoyl-CoA (Km approximately 3 microM) as substrate, kcat was 6.4 s-1. 5-HD-CoA was also a substrate for purified pig kidney MCAD. We next tested whether the reaction product, 5-hydroxydecenoyl-CoA (5-HD-enoyl-CoA), was a substrate for enoyl-CoA hydratase, the second enzyme of the beta-oxidation pathway. Similar to decenoyl-CoA, purified 5-HD-enoyl-CoA was also a substrate for the hydratase reaction. In conclusion, we have shown that 5-HD is metabolised at least as far as the third enzyme of the beta-oxidation pathway. Our results open the possibility that beta-oxidation of 5-HD or metabolic intermediates of 5-HD may be responsible for the inhibitory effects of 5-HD on preconditioning of the heart.

Inactivation of two-electron reduced medium chain acyl-CoA dehydrogenase by 2-octynoyl-CoA.

The acetylenic thioester, 2-octynoyl-CoA, inactivates medium chain acyl-CoA dehydrogenase from pig kidney by two distinct pathways depending on the redox state of the FAD prosthetic group. Inactivation of the oxidized dehydrogenase occurs with labeling of an active site glutamate residue and elimination of CoASH. Incubation of the reduced dehydrogenase with 2-octynoyl-CoA rapidly forms a kinetically stable dihydroflavin species which is resistant to reoxidation using trans-2-octenoyl-CoA, molecular oxygen, or electron transferring flavoprotein. The reduced enzyme derivative shows extensive bleaching at 446 nm with shoulders at 320 and 380 nm. Denaturation of the reduced derivative in 80% methanol yields a mixture of products which was characterized by HPLC, by uv/vis, and by radiolabeling experiments. Approximately 20% of the flavin is recovered as oxidized FAD, about 40% is retained covalently attached to the protein, and the remainder is distributed between several species eluting after FAD on reverse-phase HPLC. The spectrum of one of these species ressembles that of a N(5)-C(4a) dihydroflavin adduct. These data suggest that a primary reduced flavin species undergoes various rearrangements during release from the protein. The possibility that the inactive modified enzyme represents a covalent adduct between 2-octynoyl-CoA and reduced flavin is discussed. Analogous experiments using enzyme substituted with 1,5-dihydro-5-deaza-FAD show rapid and quantitative reoxidation of the flavin by 0.5 eq of 2-octynoyl-CoA.